Design of Molecularly Imprinted Polymer Nanoparticles Capable of Suppressing TEM-1 β-Lactamase Activity

Design of Molecularly Imprinted Polymer Nanoparticles Capable of Suppressing TEM-1 β-Lactamase Activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Design of Molecularly Imprinted Polymer Nanoparticles Capable of Suppressing TEM-1 β-Lactamase Activity Ammar Ibrahim, Alvaro Garcia-Cruz, Vian Saleh, Ameen Alrajhi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8721892/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Antimicrobial resistance (AMR) is a major global health threat, as classified by the World Health Organisation (WHO), and is driven by the ability of microorganisms to deactivate drugs. This research aims to address AMR by employing molecularly imprinted polymer nanoparticles (nano-MIPs) capable of inhibiting TEM-1 β-lactamase enzyme, a key contributor to bacterial drug resistance. We used a snapshot imprinting technique to map the epitopes of the TEM-1 enzyme. This mapping identified five crucial epitopes, which were then used to synthesize the nano-MIPs via a solid-phase protocol. The resulting nano-MIPs with 169-242 nm diameter, demonstrated exceptional affinity and selectivity for TEM-1, with dissociation constants (K D ) as low as 0.006–0.35 nM. In vitro assays confirmed the effectiveness of the nano-MIPs in inhibiting 93% of the TEM-1 activity. We also investigated antibiotic resistance in both E. coli pET15b cell cultures and the culture supernatant, where TEM-1 is released. Initial tests revealed significant resistance to ampicillin in the culture supernatant, which nano-MIPs successfully mitigated. The nanoparticles were able to reduce the effective dose of the antibiotic from 31 µg×mL -1 to 15 µg×mL -1 . The high efficiency of nano-MIPs suggests that this approach could be broadly applied to target other critical biomarkers involved in bacterial survival, offering a promising new strategy to combat AMR. Polymer Science Nanoscience Analytical Biochemistry Chemical Biology Molecularly Imprinted Polymer nanoparticles (nano-MIPs) Antimicrobial Resistance TEM-1 β-lactamase Snapshot Imprinting Enzyme Inhibition. Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction The discovery of antibiotics over the last 100 years has revolutionised the treatment of bacterial infections. The first antibiotic, salvarsan, was discovered in 1909 for syphilis treatment by Paul Ehrlich, while penicillin, the most well-known antibiotic, was discovered in the 1940s by Alexander Fleming. Fleming himself predicted that the overuse of penicillin could lead to antimicrobial resistance (AMR), 1, 2 a phenomenon that has since become a global health crisis. AMR is the ability of bacteria to develop defences against antibiotics. This has been observed in relation to almost every new antibiotic. Bacteria employ various resistance mechanisms, including the modification of penicillin-binding proteins, 3 decreased drug permeability 4 , the use of efflux pumps, 5 and enzymatic drug inactivation. 6 The World Health Organisation (WHO) classified AMR as one of the three most dangerous global health threats in 2010, 7 and it is now often referred to as the "Silent Pandemic". 8 According to the Centres for Disease Control and Prevention ( https://www.cdc.gov/ ), AMR was linked to 5 million deaths worldwide in 2019, with projections of up to 10 million deaths annually by 2050. This crisis also places a massive financial burden on healthcare systems, with estimated annual treatment costs of around 9 billion euros in Europe and $35 billion in the United States. 9 Despite the development of various new strategies, bacteria continue to evolve resistance, creating a significant challenge for healthcare organisations. β-Lactam antibiotics, which include penicillin, cephalosporins, and carbapenems, are the most widely used class of antibiotics. They are characterised by a β-lactam ring, which is essential for their bactericidal activity. A major cause of resistance to these drugs is the production of β-lactamases, enzymes found in both Gram-positive and Gram-negative bacteria that deactivate β-lactam antibiotics by cleaving this critical ring. 2, 10-12 TEM-1 β-lactamase is a Class A β-lactamase with a serine70 active site, known for its ability to hydrolyse the β-lactam ring and confer resistance to novel penicillin and cephalosporin drugs. 13 The active site of the enzyme is located within a groove and is surrounded by several crucial structural elements. These include the Ω-loop (residues 172–179), which is positioned at the active site's entrance and, along with Glu166, is essential for maintaining the active-site architecture and its role in the hydrolysis reaction. 14 Additionally, the α-3 and α-4 helices are located near the active site's borders and contain Tyrosine-105, which is important for substrate recognition. 15, 16 The α-11 and α-12 helices are also present, which include the hinge region and the allosteric site that is sandwiched between them. 17, 18 In Gram-negative bacteria, the mechanism of β-lactam antibiotic hydrolysis occurs in the periplasmic space after β-lactam molecules pass through the outer membrane. However, recent studies have shown that β-lactamases can also be secreted from Gram-negative bacteria via outer membrane vesicles (OMVs), conferring drug resistance in the extracellular environment. 19, 20 The complexity of β-lactamase structure and its various resistance mechanisms necessitates a highly specific and stable inhibitory tool. Thus, researchers have turned to advanced molecular engineering techniques. One such promising approach is molecularimprinting, a technique pioneered by Wulff and Sarhan 21 in the 1970s and further developed by Klaus Mosbach 22 in 1981. Molecularly imprinted polymers (MIPs) are synthetic materials with recognition properties similar to antibodies. MIPs are synthesised through the copolymerisation of functional monomers, a cross-linker, and a template molecule in a porogenic solvent in the presence of a template. Removal of the template creates specific binding sites in the polymer network stabilised by crosslinking. 23 When synthesised on a nanoscale (nano-MIPs), these particles exhibit high selectivity, stability, biocompatibility, and low production costs, making them ideal for a wide range of chemical and biological applications. 24-27 Our team has made significant advancements in this field, developing a solid-phase approach for synthesising nano-MIPs 28, 29 . We also introduced "snapshot imprinting," a method for identifying protein biomarkers and epitopes, which can then be used as templates for synthesis of nano-MIPs for targeting proteins. 30-32 Snapshot imprinting is a technique for epitope mapping, as shown in Fig. 1 . In this process, molecular imprinting is used to capture specific epitopes, after which the unbound portion of the protein is enzymatically cleaved. The remaining epitope is then eluted and analysed using mass spectrometry. In this study, we applied snapshot imprinting to identify crucial peptide sequences of TEM-1 β-lactamase that are related to its catalytic activity. These fragments were then analysed using liquid chromatography coupled with electrospray ionisation (ESI) and tandem mass spectrometry (LC-MS-MS). We compared the identified peptide sequences with the full TEM-1 sequence and structure from the Uniprot ( www.uniprot.org ) 33 and Protein Data Bank (PDB) ( www.rcsb.org ) 34 to confirm that they correspond to the enzyme's critical regions—specifically, the Ω-loop, the α−3 and α−4 helices, and the allosteric site—that influence its catalytic activity. The mapped epitopes were used as templates to synthesise nano-MIPs. We then evaluated the inhibitory effects of these nano-MIPs on TEM-1 activity both in vitro and in cell culture supernatant to explore their potential for tackling TEM-1 activity beyond the bacterial outer membrane. 2. Materials and methods 2.1. Enzyme structure and purity The purity and molecular weight of the enzyme were confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein sequencing and identification were performed using mass spectrometry, and data analysis was performed using Scaffold software viewer 5.3.0. The results of this experiment were added to ( Supporting information ) (Fig. S4) . 2.2. Epitope mapping of TEM-1 β-lactamase using snapshot imprinting For the mapping of TEM-1 β-lactamase, the enzyme was added to an acrylamide monomer mixture , which was then used to synthesise nano-MIPs. For that purpose, the following monomers were dissolved in ultrapure water: N-isopropyl acrylamide (NIPAM) (20 mg, 180 µmol), N-tert-butylacrylamide (TBAM) (16.5 mg, 130 µmol, dissolved in 1 mL of ethanol), N,N′-methylenebisacrylamide (BIS) (3 mg, 20 µmol), N-(3-aminopropyl) methacrylamide hydrochloride (3 mg, 17 µmol), and acrylic acid (1.1 µL, 16 µmol). After sonicating mixture for 5 min, the solution was then purged with N 2 for 20 min, and the polymerisation initiated by adding potassium persulfate (KPS) (30 mg, 0.13 mmol) and N,N,N',N'-tetramethyl ethylenediamine (TEMED) (30 µL, 0.2 mmol). Afterwards, the centrifuged mixture was resuspended with 200 µL of freshly prepared 0.1 mg×mL -1 trypsin dissolved in phosphate buffer saline (100 mM PBS) and incubated for 72 h at room temperature. After incubation, the digested peptides and trypsin were removed by centrifugation. Then, the peptides bound to nano-MIPs were removed by hot water elution. The mapped peptide concentration was determined using O-phthalaldehyde (OPA) assay 35 and identified using liquid chromatography with electrospray ionisation and tandem mass spectrometry (LC- ESI / MS-MS) ( Supporting information ). 2.3. Nano-MIPs synthesis using solid-phase protocol Nano-MIPs protocol was adopted from Piletsky et al. 29 .Glass beads (60 g, 50-150 µm) were activated by boiling in 1 M NaOH. After drying, 4% 3-iodopropyltrimethoxysilane (IPTMS) in dry toluene was added for salinisation. Afterwards, TEM-1 β-lactamase or the mapped peptides ( Table. S2) were used for immobilisation onto silanised glass beads. Then the monomeric mixture was added to the glass beads with immobilised template and KPS 30 mg×mL -1 (500 µL) and TEMED (30 µL, 0.2 mmol) were added to initiate the polymerisation reaction. After 1 hour, the glass beads with polymerisation mixture was transferred to syringe filter 20 µm and eluted with water (5 x100 mL) using vacuum to remove unreacted monomers and low-affinity MIPs. Finally, hot ethanol (65 °C, 5 x 20 mL) was used to elute nano-MIPs and concentrated to 5 mL using a vacuum evaporator. Then, using 10 kDa Snake SkinTM Dialysis, the synthesised nano-MIPs were dialysed in 1.8 L DI water for 72 h, with regular change of water. For the control, a non-imprinted polymer (NIP) was prepared following the identical procedure, except that bovine serum albumin (BSA) was coupled to the glass beads instead of the target molecule. 2.4. Characterisation of nano-MIPs The synthesised nano-MIPs were characterised using several techniques. Fourier Transform Infrared Spectroscopy (FTIR) with a Bruker Alpha platinumed FTIR spectrometer was used to determine their chemical structure. Particle size was measured by a Zeta-sizer Nano (Nano-S) from Malvern Instruments using Dynamic Light Scattering (DLS). For imaging, we used both a JEOL JEM-1400 Transmission Electron Microscope (TEM) and a Zeiss Gemini 360 FEG-SEM (Scanning Electron Microscope). Finally, the concentration of the nano-MIPs was determined by measuring the UV absorbance of serial dilutions at a wavelength of 197 nm, using a Shimadzu UV-1800 spectrophotometer (see Supporting information) . 2.5. Analysis of binding kinetics using Octet® BLI Bio-Layer Interferometry (BLI) provides a label-free analytical approach for fast and accurate measurement of biomolecular interactions, including affinity and kinetics. 36 In this study, the strength of the interaction between our nano-MIPs and their target was assessed by determining the dissociation constant (K D ) using an Octet® R8 Bio-Layer Interferometry system with AR2G biosensors. The experimental protocol for the Octet® BLI was adapted from the AR2G biosensors kit technical note from Sartorius 37 . ( Supporting information , Fig. S2) . 2.6. Assessing Nano-MIPs ability to inhibit enzyme activity The activity and inhibitory potency of β-lactamase is measured by monitoring the colour change that occurs when the enzyme hydrolyses the amide bond in chromogenic substances containing β-lactam rings. In this study, we measured the activity and inhibition of TEM-1 β-lactamase using an automated method that detects the change in absorbance of the substrate, nitrocefin, at 490 nm following hydrolysis of its β-lactam ring. Before assessing inhibition, we first confirmed the enzyme stability under the assay conditions to ensure it did not degrade. We tested enzyme activity by adding varying concentrations of TEM-1 to 40 µM of the nitrocefin substrate in a 96-well plate. To check its stability, TEM-1 at concentrations of 1, 2, and 4 nM was dissolved in 100 mM PBS containing 0.005% Triton X-100. Then, the enzyme activity was monitored by measuring the absorbance at 490 nm every 3 min, both before and after a 90-min incubation at room temperature and 37 °C. To assess the relative inhibition of TEM-1 β-lactamase, we prepared different concentrations of the synthesized nano-MIPs by dissolving them in phosphate buffer (pH 7.2) and sonicating the solution for 8 min. These nano-MIP concentrations were then incubated with 2 nM of the enzyme for 1 h at both room temperature and 37 °C. Following this incubation, 40 µM of nitrocefin was added to the 96-well plate, and the UV absorbance was immediately measured over a 60-min period. The percentage of TEM-1 β-lactamase inhibition was calculated using the following relation, Inhibition % = (ABS 2− ABS 1 ) / (T 2 −T 1 ) = Δ ABS / time (min). To quantify the effectiveness of an inhibitor, the relative inhibition percentage was calculated, as shown in Equation (1). The calculation compares the rate of enzyme activity in a control sample (Slope EC), which contains only the enzyme, to the rate of activity in a sample with the nano-MIPs present (Slope SMIP). The resulting value represents the percentage reduction in enzyme activity attributed to the inhibitor's effect. …………….. (1) 2.7 Determination of Minimum Inhibitory Concentration (MIC) for ampicillin resistance An E. coli DH5α cell culture, containing the AmpR gene on a pET15b plasmid 38 that produces TEM-1 β-lactamase, was prepared by inoculating 5 mL of Luria-Bertani (LB) media with 100 μg×mL -1 of ampicillin. To assess ampicillin resistance, we determined the Minimum Inhibitory Concentration (MIC) for both the cell culture and the culture supernatant, as detailed in the (Supporting information , Fig. S3 ). The antibacterial activity of the nano-MIPs was then studied against both the E. coli pET15b cell culture and its culture supernatant (CSpET15b) (Fig. S4) . For the cell culture experiment, nano-MIPs were prepared at concentrations of 0.1 and 0.05 mg×mL -1 in sterile 5 mM PBS buffer (pH 7.2), sonicated, and mixed with 100 μL of the E. coli pET15b cell culture (OD 580 nm = 0.1 and 4.5 x10 10 CFU × mL -1 ). The following day, we tested the growth of E. coli in each mixture using both MIC assays and agar plates to monitor the antibacterial activity of the nano-MIPs. For the supernatant experiment, the E. coli pET15b cell culture was centrifuged, and the cell pellet was discarded. The supernatant (CSpET15b) was then filtered using a 0.2 μm syringe filter. This filtered supernatant was incubated with 0.05 mg×mL -1 of each prepared nano-MIP, dissolved in 5 mM PBS, pH 7.2 at 37°C for 24 hours. 3. Results and discussion 3.1. Mapping TEM-1 β-lactamase epitopes with nano-MIPs In snapshot imprinting, once polymerisation was complete, the peptides were eluted and separated from the nano-MIPs via centrifugation. The hydrodynamic size of the resulting nano-MIPs was measured using DLS, which yielded an average particle size of 194.7±114 nm. TEM images confirmed the DLS results, showing spherical and semi-spherical particles with a size distribution comparable to the DLS data ( Fig. S6 ). The concentration of the mapped peptides was found to be 127.5 μg×mL -1 , which is sufficient for the sequencing experiment. Peptide analysis was performed using Progenesis QI for Proteomics (version 4.2), which identified 125 sequences. Of these, 51 sequences were highly confident (scores 4-7) and were frequently reported. Based on the normalised abundance and scores from the LC-MS-MS report, 99.99% of the protein structure was successfully mapped, including regions critical to catalytic activity ( Fig. 2), (Table S3). Based on existing literature, five specific epitopes known to contribute to the structural dynamics and catalytic activity of TEM-1 β-lactamase were selected as templates for further nano-MIP synthesis as shown in Table 1 . 14-18 This table details the specific peptide sequences chosen as templates for the synthesis of nano-MIPs. To enable their immobilisation on glass beads for the solid-phase synthesis of nano-MIPs, a terminal cysteine was added to each peptide sequence. This allowed for a stable reaction with the iodoalkyl ends on the IPTMS-silanised glass bead surface. (Table 1). Epitopes selected as templates for nano-MIPs synthesis Residues Template Sequence Domain Notes CQQLIDWMEADK EP-1 203 - 213 Participate in the hinge region α 11 Allosteric site CLNEAIPNDERDTT EP-2 166-178 Ω – loop & Glu 166 - Asn 172 - CTTDREDNPIAENL EP-3 178-166 Ω – loop & Glu 166 - Asn 172 Scramble CRIHYSQNDLVEYSPVTE EP-4 92-108 α 11 and α 12 -Tyrosine 105 - CMEADKVAGPLLRS EP-5 209-221 α 11 Allosteric site 3.2. Characterisation of synthesised nano-MIPs Based on the mapped peptides, nano-MIPs were successfully synthesised. The particle sizes of these nano-MIPs, as measured by DLS, ranged from 169 to 242 nm ( Table S6, Fig. S9 ). Electron microscopy images from both TEM and SEM further confirmed these DLS measurements, with particle diameters and standard deviations showing high consistency. In some TEM images, nano-MIPs appeared as clustered semi-spherical particles ( Fig. S10 ), which could be a result of particle aggregation caused by drying. Most of TEM and SEM images revealed the nano-MIPs as discrete, single spherical particles. The final concentration of the nano-MIPs was determined by gravimetric analysis after lyophilisation and by a spectroscopic method using UV absorbance, as detailed in Fig. S11 . 3.3. Analysis of binding kinetics using Octet® BLI The optimal assay condition for nano-MIP binding with TEM-1 was at pH 5.0 where better association signal was observed ( Fig. S12) . All the synthesised nano-MIPs showed significant affinity and quick saturation (500-600) seconds with TEM-1 β-lactamase ( Table S4) . The association curves showed clear binding signals between nano-MIPs and the enzyme ( Fig. S12 ), and the affinity of nano-MIPs prepared by imprinting of the mapped epitopes was higher in comparison with nano-MIP@TEM-1, this result supports the efficiency of epitope mapping and epitope imprinting. Furthermore, K D values of all synthesised nano-MIPs were lower than NIP (K D = 28 nM). It is powerful evidence that confirms the selectivity of the synthesised nano-MIPs. 3.4. Assessment of enzyme activity and stability of TEM-1 β-lactamase The enzyme showed clear increase in the UV absorbance of 40 µM nitrocefin over time at 490 nm. Higher concentrations of TEM-1 showed very fast colour change and absorbance increased very quickly. This confirms the enzyme activity towards the substrate. With lower concentrations, the colour changed gradually; therefore, 2 nM of the enzyme was considered for the assay with nano-MIPs (Fig. S13) . Also, the enzyme remained stable and showed similar absorbance curves at the assay conditions, including room temperature and 37 o C (Fig. S14) . This result is in agreement with Grigorenko et al. 39 , who demonstrated TEM-1 activity at the broad temperature range (41 – 57) °C. 3.5. Inhibition of TEM-1 activity using nano-MIPs Significant reductions in enzyme activity were observed in the presence of nano-MIPs ( Fig. S15 ), indicating enzyme inhibition. While TEM-1 β-lactamase is known for its rigid and conserved structural architecture, its catalytic activity can be significantly altered when a ligand binds to a specific region, potentially blocking the active site. Our results demonstrate that nano-MIPs, synthesised using key epitopes (EP-1 and EP-5 from the allosteric site, EP-2 from the Ω-loop, and EP-4 from the α3-α4 region, including Tyr105), effectively inhibit the enzyme. This inhibition is consistent with the hypothesis that ligand interactions with the allosteric site (α11-α12) can induce dynamic conformational changes in surrounding loops, such as the Ω-loop (residues 172-179). The Ω-loop plays a crucial role in the catalytic hydrolysis of the β-lactam ring; its fluctuation or rotation, initiated from the allosteric site, can suppress its catalytic enhancement role. 40-42 Quantitatively, at 20 °C, nano-MIP@TEM-1, nano-MIP@EP-1, and nano-MIP@EP-2 exhibited potent inhibition, exceeding 90% of enzyme activity reduction at concentrations ranging from 1 to 22 pM. Nano-MIP@EP-3, nano-MIP@EP-4, and nano-MIP@EP-5 also showed substantial inhibition, achieving 85%, 79%, and 47% respectively. Notably, at 37°C, all synthesised nano-MIPs demonstrated even higher efficacy, inhibiting more than 93% of the enzyme activity ( Table 2 ). Table 2. Enzyme inhibition percentage (>90%) by nano-MIPs. 20C ο 37C ο Nano-MIP@ TEM-1 % inhibition 94 ±3.8 93 ± 4.0 Concentration, pM 1.11 11.08 Nano-MIP@EP-1 % inhibition 97 ± 2.0 94 ± 4.7 Concentration, pM 22.2 1.44 Nano-MIP@EP-2 % inhibition 99 ± 1.0 99 ± 0.35 Concentration, pM 1.11 0.22 Nano-MIP@EP-3 % inhibition 85 ± 3.31 99 ± 0.98 Concentration, pM 45.8 92.34 Nano-MIP@EP-4 % inhibition 79 ± 2.77 93.57 ± 0.67 Concentration, pM 184 369 Nano-MIP@EP-5 % inhibition 47.6 ± 7.2 93 ± 1.4 Concentration, pM 184 2954 NIP % inhibition N/A N/A Concentration, pM N/A N/A The improved efficiency of nano-MIPs against enzyme activity at 37°C can be attributed to higher thermal energy, which enhances both the motion of nano-MIPs and the enzyme, thereby increasing binding by improving the orientational susceptibility of nano-MIP binding sites and imprinted regions. Interestingly, some nano-MIPs demonstrated enzyme inhibition even at low nanoparticles concentrations. Higher concentrations of nano-MIPs in general lead to a stronger inhibitory effect, ( Fig. S16, Fig. S17 ), similar to other antibacterial β-lactamases inhibitors. 43-45 Some deviation from linearity of the response might be explained by aggregation of nanoparticles at room temperature. Nano-MIPs at 37°C showed a clear inhibition dose response across a wide concentration range. The evidence of imprinting effect can be seen from the BLI and enzyme inhibition results obtained for nano-MIP@EP-3. This nano-MIP was synthesised by imprinting EP-3, a scrambled version of EP-2 (with a reversed amino acid sequence). The affinity of nano-MIP@EP-3 (K D = 0.18 nM) to enzyme was 30 times lower than that of nano-MIP@EP-2 (K D = 0.006 nM). These findings are corroborated by the enzyme inhibition data, where nano-MIP@EP-2 demonstrated a superior inhibitory effect compared to nano-MIP@EP-3. The MIC results demonstrated that 6.3 µg×mL -1 of ampicillin was the minimum concentration required to suppress E. coli pET15b growth. The ampicillin MIC assay in CSpET15b revealed clear evidence of AMR: the wild-type (WT) control grew in all CSpET15b wells, whereas it was suppressed by ampicillin in LB medium, and in CSWT at 6.3 µg.mL -1 ( Fig. S18 ). The Resazurin dye colour change confirmed that WT cells maintained similar viability levels in both WT cell culture and CSWT. Crucially, WT cells continued to grow at all ampicillin concentrations when CSpET15b was present, unequivocally demonstrating the existence of TEM-1 β-lactamase within CSpET15b and its impact on AMR. This finding strongly suggests that TEM-1 β-lactamase is released into the external milieu from bacteria ( Fig. S18 ). The results of the disk diffusion filter test further confirmed the ampicillin resistance conferred by the E. coli pET15b culture supernatant (CSpET15b). As shown in Fig. S20 , the growth of ampicillin-sensitive wild-type ( E. coli WT) bacteria around the filter disk loaded with CSpET15b demonstrated that the ampicillin in the agar was degraded by the secreted TEM-1 β-lactamase enzyme. This was in contrast to the control, where the CSWT (which lacks the enzyme) failed to degrade the ampicillin, which inhibited the growth of the WT strain. The antibiotic resistance of the culture supernatant was shown to be concentration-dependent, as reduction in the concentration of the CSpET15b led to a decrease in its ability to protect the WT strain from ampicillin ( Fig. S19 ). However, an interesting observation was made when WT strains were exposed to different concentrations of CSpET15b. While WT bacteria exposed to diluted CSpET15b (10%, 1%, 0.1%, and 0.05%) did not grow on ampicillin-agar plates, the WT bacteria exposed to 100% CSpET15b did exhibit resistance. This suggests that the high concentration of TEM-1 β-lactamase in the undiluted supernatant was sufficient to degrade a large amount of ampicillin. Alternatively, this result could be attributed to the transfer of the pET15b plasmid from the culture supernatant to the WT E. coli cells, a process known as horizontal gene transfer ( Fig. S21 ). These findings provide compelling evidence for the release of TEM-1 β-lactamase into the extracellular environment, a phenomenon that is not yet fully verified for all Gram-negative bacteria. While most research suggests that β-lactamases are confined to the periplasmic space or are membrane-bound, some studies, such as that by Schaar et al. 19 , have shown that Gram-negative bacteria like Moraxella catarrhalis can secrete these enzymes packaged within outer membrane vesicles (OMVs). 19 , 46 This mechanism allows them to inactivate antibiotics and promote the survival of other bacteria. To some extent, the results of this experiment offer new perspectives on the development of antibiotic resistance and highlight the potential for extracellular TEM-1 β-lactamase to play a role in this process. The activity of the synthesized nano-MIPs was evaluated in both, presence and absence of bacterial cells. When incubated with live E. coli pET15b cell cultures, the nano-MIPs were unable to suppress antibiotic resistance ( Fig. 3 ). This lack of effect is probably due to the inability of the nano-MIPs to diffuse through the bacterial outer membrane and into the periplasmic space where the TEM-1 β-lactamase is located. Alternatively, the high concentration of the enzyme in the medium may have saturated the limited binding capacity of the nano-MIPs. However, in a supernatant the nano-MIPs demonstrated a clear inhibitory effect. After an overnight incubation with the culture supernatant (CSpET15b), both Nano-MIP@TEM-1 and Nano-MIP@EP-2 were able to reduce the ampicillin resistance of the solution. The minimum inhibitory concentration (MIC) for ampicillin was lowered from 31 µg×mL -1 to 15 µg×mL -1 ( Fig. 4 ), demonstrating the ability of the nano-MIPs to effectively suppress the TEM-1 β-lactamase enzyme activity in the supernatant. 4. Conclusion The increasing capacity of bacteria to deactivate antibiotics poses a major threat to public health and the economy. In this work, we explored the use of nano-MIPs as synthetic alternatives to biological receptors for targeting the TEM-1 β-lactamase enzyme, a key mediator of antibiotic resistance. A novel snapshot imprinting method was used to identify effective binding domains for nano-MIPs on the protein structure. Crucially, this included the identification of domains known to be connected to the enzyme's active site (Ser70), such as the α3-α4 loop, the Ω-loop, and the allosteric site. Five of these mapped epitopes were selected as templates for synthesising nano-MIPs using a solid-phase protocol. The resulting nano-MIPs were spherical, with particle sizes ranging from 169 to 242 nm. The binding kinetics of these nano-MIPs were evaluated using Octet® Biolayer Interferometry (BLI). The data showed that the nano-MIPs possessed excellent affinity and selectivity for TEM-1, with picomolar-scale affinity constants (K D ). Furthermore, a functional assay demonstrated that the nano-MIPs were able to inhibit more than 93–99% of the enzyme's activity. We hypothesise that this inhibition is a result of the nano-MIPs binding to key domains on the enzyme, which disturbs its conformational dynamics and prevents the antibiotic substrate from accessing the active site. To investigate the phenomenon of TEM-1 β-lactamase release, we studied the antibiotic resistance of E. coli pET15b in both the cell culture and its supernatant. The results indicated that the culture supernatant conferred an antibiotic resistance level equivalent to that of the live cell culture. This finding was further supported by a disk diffusion test and a titration of the supernatant's antibiotic resistance, providing evidence that TEM-1 β-lactamase is released beyond the bacterial outer membrane. When tested against live bacterial cultures, the nano-MIPs were ineffective at suppressing antibiotic resistance. This is likely due to their inability to diffuse into the periplasmic space to interact with the enzyme. However, in the cell-free culture supernatant, the nano-MIPs successfully suppressed AMR, reducing the minimum inhibitory concentration (MIC) of ampicillin from 31 µg×mL -1 to 15 µg×mL -1 . 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Shoichet, Journal of molecular biology , 2004, 336 , 1283-1291. V. Schaar, T. Nordström, M. Mörgelin and K. Riesbeck, Antimicrobial agents and chemotherapy , 2011, 55 , 3845-3853. L. Capodimonte, F. T. P. Meireles, G. Bahr, R. A. Bonomo, M. Dal Peraro, C. López and A. J. Vila, mBio , 2025, 16 , e03343-03324. G. Wuff and A. Sarhan, Angew. Chem. Int. Ed , 1972, 11 , 341-345. R. Arshady and K. Mosbach, Die Makromolekulare Chemie: Macromolecular Chemistry and Physics , 1981, 182 , 687-692. M. Włoch and J. Datta, in Comprehensive analytical chemistry , Elsevier, 2019, vol. 86, pp. 17-40. J. Wackerlig and P. A. Lieberzeit, Sensors and Actuators B: Chemical , 2015, 207 , 144-157. S. A. Piletsky, N. W. Turner and P. Laitenberger, Medical engineering & physics , 2006, 28 , 971-977. W. Li and S. Li, 2007. B. H. Abd, University of Leicester, 2018. F. Canfarotta, A. Poma, A. Guerreiro and S. Piletsky, Nature Protocols , 2016, 11 , 443-455. S. S. Piletsky, A. Garcia Cruz, E. Piletska, S. A. Piletsky, E. O. Aboagye and A. C. Spivey, Polymers , 2022, 14 , 1595. S. S. Piletsky, E. Piletska, M. Poblocka, S. Macip, D. J. Jones, M. Braga, T. H. Cao, R. Singh, A. C. Spivey and E. O. Aboagye, Nano Today , 2021, 41 , 101304. E. Piletska, D. Thompson, R. Jones, A. G. Cruz, M. Poblocka, F. Canfarotta, R. Norman, S. Macip, D. J. Jones and S. Piletsky, Nanoscale Advances , 2022, 4 , 5304-5311. S. A. Piletsky, T. S. Bedwell, R. Paoletti, K. Karim, F. Canfarotta, R. Norman, D. J. Jones, N. W. Turner and E. V. Piletska, Journal of Materials Chemistry B , 2022, 10 , 6732-6741. Uniprot. P. D. Bank. F. C. Church, D. H. Porter, G. L. Catignani and H. E. Swaisgood, Analytical biochemistry , 1985, 146 , 343-348. R. L. Rich and D. G. Myszka, Analytical biochemistry , 2006, 361 , 1-6. Sartorius. SnapGen. V. G. Grigorenko, A. V. Krivitskaya, M. G. Khrenova, M. Y. Rubtsova, G. V. Presnova, I. P. Andreeva, O. V. Serova and A. M. Egorov, International Journal of Molecular Sciences , 2024, 25 , 7691. I. Galdadas, S. Qu, A. S. F. Oliveira, E. Olehnovics, A. R. Mack, M. F. Mojica, P. K. Agarwal, C. L. Tooke, F. L. Gervasio and J. Spencer, Elife , 2021, 10 , e66567. C. Avery, L. Baker and D. J. Jacobs, Entropy , 2022, 24 , 729. O. Fisette, S. Gagné and P. Lagüe, Biophysical journal , 2012, 103 , 1790-1801. T. R. J. Mary, R. R. Kannan, A. M. Iniyan, W. A. C. Ranjith, S. Nandhagopal, V. Vishwakarma and S. G. P. Vincent, Microbiological Research , 2021, 244 , 126666. F. Ahmad, N. Parvaiz, A. D. MacKerell Jr and S. S. Azam, Journal of Chemical Information and Modeling , 2023, 63 , 6681-6695. A. Ali, Danishuddin, L. Maryam, G. Srivastava, A. Sharma and A. U. Khan, Journal of Biomolecular Structure and Dynamics , 2018, 36 , 1806-1821. X. Zeng and J. Lin, Frontiers in microbiology , 2013, 4 , 128. Additional Declarations The authors declare no competing interests. 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Labelled and visualised by PyMole molecular graphic system v.3.0.3 (Left), and UCSF Chimera v1.17.3 (Right). This figure illustrates the distinct regions of the TEM-1 β-lactamase protein, colour-coded for clarity: Unmapped sequence (Blue), Peptide sequence 1-118 (Red), Peptide sequence 119-190 (Yellow), and Peptide sequence 191-286 (Green). b) Selected epitopes (from the effective regions) as templates for nano-MIPs synthesis\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8721892/v1/4e6683e734193a14783e62f3.png"},{"id":101845362,"identity":"bbf4732e-cf86-47e5-b206-e7ad13b312d3","added_by":"auto","created_at":"2026-02-04 09:12:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":137400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eThe optical density of E.coli pET15b growth in the presence of serial dilutions of ampicillin after incubation with nano-MIPs overnight.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8721892/v1/79d327396c6907f0837a05ce.png"},{"id":101845432,"identity":"8b18676d-8899-4d76-95cb-c2c5e371cd54","added_by":"auto","created_at":"2026-02-04 09:12:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":76135,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEffect of nano-MIPs in reducing MIC in CSpET15b. (SD±0.00028-0.027)\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8721892/v1/6bb5bbc5c0691608c4808f21.png"},{"id":101845450,"identity":"e7b95dc1-d7ed-4d06-8a3f-23e9532adf0f","added_by":"auto","created_at":"2026-02-04 09:12:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2028925,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8721892/v1/ed9d4b50-953f-4302-9e5e-ed7e9fa0aafc.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eDesign of Molecularly Imprinted Polymer Nanoparticles Capable of Suppressing TEM-1 β-Lactamase Activity\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe discovery of antibiotics over the last 100 years has revolutionised the treatment of bacterial infections. The first antibiotic, salvarsan, was discovered in 1909 for syphilis treatment by Paul Ehrlich, while penicillin, the most well-known antibiotic, was discovered in the 1940s by Alexander Fleming. Fleming himself predicted that the overuse of penicillin could lead to antimicrobial resistance (AMR),\u003csup\u003e1, 2\u003c/sup\u003e a phenomenon that has since become a global health crisis. AMR is the ability of bacteria to develop defences against antibiotics. This has been observed in relation to almost every new antibiotic. Bacteria employ various resistance mechanisms, including the modification of penicillin-binding proteins,\u003csup\u003e3\u003c/sup\u003e decreased drug permeability\u003csup\u003e4\u003c/sup\u003e, the use of efflux pumps,\u003csup\u003e5\u003c/sup\u003e and enzymatic drug inactivation.\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eThe World Health Organisation (WHO) classified AMR as one of the three most dangerous global health threats in 2010,\u003csup\u003e7\u003c/sup\u003e and it is now often referred to as the \u0026quot;Silent Pandemic\u0026quot;.\u003csup\u003e8\u003c/sup\u003e According to the Centres for Disease Control and Prevention (\u003cstrong\u003ehttps://www.cdc.gov/\u003c/strong\u003e), AMR was linked to 5 million deaths worldwide in 2019, with projections of up to 10 million deaths annually by 2050. This crisis also places a massive financial burden on healthcare systems, with estimated annual treatment costs of around 9 billion euros in Europe and $35 billion in the United States.\u003csup\u003e9\u003c/sup\u003e Despite the development of various new strategies, bacteria continue to evolve resistance, creating a significant challenge for healthcare organisations. \u003c/p\u003e\n\u003cp\u003e\u0026beta;-Lactam antibiotics, which include penicillin, cephalosporins, and carbapenems, are the most widely used class of antibiotics. They are characterised by a \u0026beta;-lactam ring, which is essential for their bactericidal activity. A major cause of resistance to these drugs is the production of \u0026beta;-lactamases, enzymes found in both Gram-positive and Gram-negative bacteria that deactivate \u0026beta;-lactam antibiotics by cleaving this critical ring.\u003csup\u003e2, 10-12\u003c/sup\u003e TEM-1 \u0026beta;-lactamase is a Class A \u0026beta;-lactamase with a serine70 active site, known for its ability to hydrolyse the \u0026beta;-lactam ring and confer resistance to novel penicillin and cephalosporin drugs.\u003csup\u003e13\u003c/sup\u003e The active site of the enzyme is located within a groove and is surrounded by several crucial structural elements. These include the \u0026Omega;-loop (residues 172\u0026ndash;179), which is positioned at the active site\u0026apos;s entrance and, along with Glu166, is essential for maintaining the active-site architecture and its role in the hydrolysis reaction.\u003csup\u003e14\u003c/sup\u003e Additionally, the \u0026alpha;-3 and \u0026alpha;-4 helices are located near the active site\u0026apos;s borders and contain Tyrosine-105, which is important for substrate recognition.\u003csup\u003e15, 16\u003c/sup\u003e The \u0026alpha;-11 and \u0026alpha;-12 helices are also present, which include the hinge region and the allosteric site that is sandwiched between them.\u003csup\u003e17, 18\u003c/sup\u003e In Gram-negative bacteria, the mechanism of \u0026beta;-lactam antibiotic hydrolysis occurs in the periplasmic space after \u0026beta;-lactam molecules pass through the outer membrane. However, recent studies have shown that \u0026beta;-lactamases can also be secreted from Gram-negative bacteria via outer membrane vesicles (OMVs), conferring drug resistance in the extracellular environment.\u003csup\u003e19, 20\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eThe complexity of \u0026beta;-lactamase structure and its various resistance mechanisms necessitates a highly specific and stable inhibitory tool. Thus, researchers have turned to advanced molecular engineering techniques. One such promising approach is molecularimprinting, a technique pioneered by Wulff and Sarhan\u003csup\u003e21\u003c/sup\u003e in the 1970s and further developed by Klaus Mosbach\u003csup\u003e22\u003c/sup\u003e in 1981. Molecularly imprinted polymers (MIPs) are synthetic materials with recognition properties similar to antibodies. MIPs are synthesised through the copolymerisation of functional monomers, a cross-linker, and a template molecule in a porogenic solvent in the presence of a template. Removal of the template creates specific binding sites in the polymer network stabilised by crosslinking.\u003csup\u003e23\u003c/sup\u003e When synthesised on a nanoscale (nano-MIPs), these particles exhibit high selectivity, stability, biocompatibility, and low production costs, making them ideal for a wide range of chemical and biological applications.\u003csup\u003e24-27\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eOur team has made significant advancements in this field, developing a solid-phase approach for synthesising nano-MIPs\u003csup\u003e28, 29\u003c/sup\u003e. We also introduced \u0026quot;snapshot imprinting,\u0026quot; a method for identifying protein biomarkers and epitopes, which can then be used as templates for synthesis of nano-MIPs for targeting proteins.\u003csup\u003e30-32\u003c/sup\u003e Snapshot imprinting is a technique for epitope mapping, as shown in \u003cstrong\u003eFig. 1\u003c/strong\u003e. In this process, molecular imprinting is used to capture specific epitopes, after which the unbound portion of the protein is enzymatically cleaved. The remaining epitope is then eluted and analysed using mass spectrometry. In this study, we applied snapshot imprinting to identify crucial peptide sequences of TEM-1 \u0026beta;-lactamase that are related to its catalytic activity. These fragments were then analysed using liquid chromatography coupled with electrospray ionisation (ESI) and tandem mass spectrometry (LC-MS-MS). We compared the identified peptide sequences with the full TEM-1 sequence and structure from the Uniprot (\u003cstrong\u003ewww.uniprot.org\u003c/strong\u003e)\u003csup\u003e33\u003c/sup\u003e and Protein Data Bank (PDB) (\u003cstrong\u003ewww.rcsb.org\u003c/strong\u003e)\u003csup\u003e34\u003c/sup\u003e to confirm that they correspond to the enzyme\u0026apos;s critical regions\u0026mdash;specifically, the \u0026Omega;-loop, the \u0026alpha;\u0026minus;3 and \u0026alpha;\u0026minus;4 helices, and the allosteric site\u0026mdash;that influence its catalytic activity. The mapped epitopes were used as templates to synthesise nano-MIPs. We then evaluated the inhibitory effects of these nano-MIPs on TEM-1 activity both \u003cem\u003ein vitro\u003c/em\u003e and in cell culture supernatant to explore their potential for tackling TEM-1 activity beyond the bacterial outer membrane.\u003c/p\u003e"},{"header":"2.\tMaterials and methods","content":"\u003cp\u003e2.1. Enzyme structure and purity\u003c/p\u003e\n\u003cp\u003eThe purity and molecular weight of the enzyme were confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein sequencing and identification were performed using mass spectrometry, and data analysis was performed using Scaffold software viewer 5.3.0. The results of this experiment were added to (\u003cstrong\u003eSupporting information\u003c/strong\u003e)\u003cstrong\u003e (Fig. S4)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e2.2. Epitope mapping of TEM-1 \u0026beta;-lactamase using snapshot imprinting\u003c/p\u003e\n\u003cp\u003eFor the mapping of TEM-1 \u0026beta;-lactamase, the enzyme was added to an \u003cem\u003eacrylamide monomer mixture\u003c/em\u003e, which was then used to synthesise nano-MIPs. For that purpose, the following monomers were dissolved in ultrapure water: N-isopropyl acrylamide (NIPAM) (20 mg, 180 \u0026micro;mol), N-tert-butylacrylamide (TBAM) (16.5 mg, 130 \u0026micro;mol, dissolved in 1 mL of ethanol), N,N\u0026prime;-methylenebisacrylamide (BIS) (3 mg, 20 \u0026micro;mol), N-(3-aminopropyl) methacrylamide hydrochloride (3 mg, 17 \u0026micro;mol), and acrylic acid (1.1 \u0026micro;L, 16 \u0026micro;mol). After sonicating mixture for 5 min, the solution was then purged with N\u003csub\u003e2\u003c/sub\u003e for 20 min, and the polymerisation initiated by adding potassium persulfate (KPS) (30 mg, 0.13 mmol) and N,N,N\u0026apos;,N\u0026apos;-tetramethyl ethylenediamine (TEMED) (30 \u0026micro;L, 0.2 mmol). Afterwards, the centrifuged mixture was resuspended with 200 \u0026micro;L of freshly prepared 0.1 mg\u0026times;mL\u003csup\u003e-1\u003c/sup\u003e trypsin dissolved in phosphate buffer saline (100 mM PBS) and incubated for 72 h at room temperature. After incubation, the digested peptides and trypsin were removed by centrifugation. Then, the peptides bound to nano-MIPs were removed by hot water elution. The mapped peptide concentration was determined using O-phthalaldehyde (OPA) assay\u003csup\u003e35\u003c/sup\u003e and identified using liquid chromatography with electrospray ionisation and tandem mass spectrometry (LC- ESI / MS-MS) (\u003cstrong\u003eSupporting information\u003c/strong\u003e). \u003c/p\u003e\n\u003cp\u003e2.3. Nano-MIPs synthesis using solid-phase protocol\u003c/p\u003e\n\u003cp\u003eNano-MIPs protocol was adopted from Piletsky et al.\u003csup\u003e29\u003c/sup\u003e.Glass beads (60 g, 50-150 \u0026micro;m) were activated by boiling in 1 M NaOH. After drying, 4% 3-iodopropyltrimethoxysilane (IPTMS) in dry toluene was added for salinisation. Afterwards, TEM-1 \u0026beta;-lactamase or the mapped peptides (\u003cstrong\u003eTable. S2) \u003c/strong\u003ewere used for immobilisation onto silanised glass beads. Then the monomeric mixture was added to the glass beads with immobilised template and KPS 30 mg\u0026times;mL\u003csup\u003e-1\u003c/sup\u003e (500 \u0026micro;L) and TEMED (30 \u0026micro;L, 0.2 mmol) were added to initiate the polymerisation reaction. After 1 hour, the glass beads with polymerisation mixture was transferred to syringe filter 20 \u0026micro;m and eluted with water (5 x100 mL) using vacuum to remove unreacted monomers and low-affinity MIPs. Finally, hot ethanol (65 \u0026deg;C, 5 x 20 mL) was used to elute nano-MIPs and concentrated to 5 mL using a vacuum evaporator. Then, using 10 kDa Snake SkinTM Dialysis, the synthesised nano-MIPs were dialysed in 1.8 L DI water for 72 h, with regular change of water. For the control, a non-imprinted polymer (NIP) was prepared following the identical procedure, except that bovine serum albumin (BSA) was coupled to the glass beads instead of the target molecule. \u003c/p\u003e\n\u003cp\u003e2.4. Characterisation of nano-MIPs\u003c/p\u003e\n\u003cp\u003eThe synthesised nano-MIPs were characterised using several techniques. Fourier Transform Infrared Spectroscopy (FTIR) with a Bruker Alpha platinumed FTIR spectrometer was used to determine their chemical structure. Particle size was measured by a Zeta-sizer Nano (Nano-S) from Malvern Instruments using Dynamic Light Scattering (DLS). For imaging, we used both a JEOL JEM-1400 Transmission Electron Microscope (TEM) and a Zeiss Gemini 360 FEG-SEM (Scanning Electron Microscope). Finally, the concentration of the nano-MIPs was determined by measuring the UV absorbance of serial dilutions at a wavelength of 197 nm, using a Shimadzu UV-1800 spectrophotometer (see \u003cstrong\u003eSupporting information)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e2.5. Analysis of binding kinetics using Octet\u0026reg; BLI\u003c/p\u003e\n\u003cp\u003eBio-Layer Interferometry (BLI) provides a label-free analytical approach for fast and accurate measurement of biomolecular interactions, including affinity and kinetics.\u003csup\u003e36\u003c/sup\u003e In this study, the strength of the interaction between our nano-MIPs and their target was assessed by determining the dissociation constant (K\u003csub\u003eD\u003c/sub\u003e) using an Octet\u0026reg; R8 Bio-Layer Interferometry system with AR2G biosensors. The experimental protocol for the Octet\u0026reg; BLI was adapted from the AR2G biosensors kit technical note from Sartorius\u003csup\u003e37\u003c/sup\u003e. (\u003cstrong\u003eSupporting information\u003c/strong\u003e\u003cstrong\u003e, Fig. S2)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e2.6. Assessing Nano-MIPs ability to inhibit enzyme activity \u003c/p\u003e\n\u003cp\u003eThe activity and inhibitory potency of \u0026beta;-lactamase is measured by monitoring the colour change that occurs when the enzyme hydrolyses the amide bond in chromogenic substances containing \u0026beta;-lactam rings. In this study, we measured the activity and inhibition of TEM-1 \u0026beta;-lactamase using an automated method that detects the change in absorbance of the substrate, nitrocefin, at 490 nm following hydrolysis of its \u0026beta;-lactam ring. Before assessing inhibition, we first confirmed the enzyme stability under the assay conditions to ensure it did not degrade. We tested enzyme activity by adding varying concentrations of TEM-1 to 40 \u0026micro;M of the nitrocefin substrate in a 96-well plate. To check its stability, TEM-1 at concentrations of 1, 2, and 4 nM was dissolved in 100 mM PBS containing 0.005% Triton X-100. Then, the enzyme activity was monitored by measuring the absorbance at 490 nm every 3 min, both before and after a 90-min incubation at room temperature and 37 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003eTo assess the relative inhibition of TEM-1 \u0026beta;-lactamase, we prepared different concentrations of the synthesized nano-MIPs by dissolving them in phosphate buffer (pH 7.2) and sonicating the solution for 8 min. These nano-MIP concentrations were then incubated with 2 nM of the enzyme for 1 h at both room temperature and 37 \u0026deg;C. Following this incubation, 40 \u0026micro;M of nitrocefin was added to the 96-well plate, and the UV absorbance was immediately measured over a 60-min period. The percentage of TEM-1 \u0026beta;-lactamase inhibition was calculated using the following relation, Inhibition % = (ABS\u003csub\u003e2\u0026minus;\u003c/sub\u003eABS\u003csub\u003e1\u003c/sub\u003e) / (T\u003csub\u003e2\u003c/sub\u003e\u0026minus;T\u003csub\u003e1\u003c/sub\u003e) = \u0026Delta; ABS / time (min). To quantify the effectiveness of an inhibitor, the relative inhibition percentage was calculated, as shown in Equation (1). The calculation compares the rate of enzyme activity in a control sample (Slope EC), which contains only the enzyme, to the rate of activity in a sample with the nano-MIPs present (Slope SMIP). The resulting value represents the percentage reduction in enzyme activity attributed to the inhibitor\u0026apos;s effect.\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"374\" height=\"57\" 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\" alt=\"image\"\u003e \u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;.. (1)\u003c/p\u003e\n\u003cp\u003e2.7 Determination of Minimum Inhibitory Concentration (MIC) for ampicillin resistance\u003c/p\u003e\n\u003cp\u003eAn \u003cem\u003eE. coli\u003c/em\u003e DH5\u0026alpha; cell culture, containing the AmpR gene on a pET15b plasmid\u003csup\u003e38\u003c/sup\u003e that produces TEM-1 \u0026beta;-lactamase, was prepared by inoculating 5 mL of Luria-Bertani (LB) media with 100 \u0026mu;g\u0026times;mL\u003csup\u003e-1\u003c/sup\u003e of ampicillin. To assess ampicillin resistance, we determined the Minimum Inhibitory Concentration (MIC) for both the cell culture and the culture supernatant, as detailed in the \u003cstrong\u003e(Supporting information\u003c/strong\u003e\u003cstrong\u003e, Fig. S3\u003c/strong\u003e). The antibacterial activity of the nano-MIPs was then studied against both the \u003cem\u003eE. coli\u003c/em\u003e pET15b cell culture and its culture supernatant (CSpET15b) \u003cstrong\u003e(Fig. S4)\u003c/strong\u003e. For the cell culture experiment, nano-MIPs were prepared at concentrations of 0.1 and 0.05 mg\u0026times;mL\u003csup\u003e-1\u003c/sup\u003e in sterile 5 mM PBS buffer (pH 7.2), sonicated, and mixed with 100 \u0026mu;L of the \u003cem\u003eE. coli\u003c/em\u003e pET15b cell culture (OD\u003csub\u003e580 nm \u003c/sub\u003e= 0.1 and 4.5 x10\u003csup\u003e10\u003c/sup\u003e CFU \u0026times; mL\u003csup\u003e-1\u003c/sup\u003e). The following day, we tested the growth of \u003cem\u003eE. coli\u003c/em\u003e in each mixture using both MIC assays and agar plates to monitor the antibacterial activity of the nano-MIPs. For the supernatant experiment, the \u003cem\u003eE. coli\u003c/em\u003e pET15b cell culture was centrifuged, and the cell pellet was discarded. The supernatant (CSpET15b) was then filtered using a 0.2 \u0026mu;m syringe filter. This filtered supernatant was incubated with 0.05 mg\u0026times;mL\u003csup\u003e-1\u003c/sup\u003e of each prepared nano-MIP, dissolved in 5 mM PBS, pH 7.2 at 37\u0026deg;C for 24 hours.\u003c/p\u003e"},{"header":"3.\tResults and discussion","content":"\u003ch2\u003e3.1.\u0026nbsp;Mapping TEM-1 \u0026beta;-lactamase epitopes with nano-MIPs\u003c/h2\u003e\n\u003cp\u003eIn snapshot imprinting, once polymerisation was complete, the peptides were eluted and separated from the nano-MIPs via centrifugation. The hydrodynamic size of the resulting nano-MIPs was measured using DLS, which yielded an average particle size of 194.7\u0026plusmn;114 nm. TEM images confirmed the DLS results, showing spherical and semi-spherical particles with a size distribution comparable to the DLS data (\u003cstrong\u003eFig. S6\u003c/strong\u003e). The concentration of the mapped peptides was found to be 127.5 \u0026mu;g\u0026times;mL\u003csup\u003e-1\u003c/sup\u003e, which is sufficient for the sequencing experiment. Peptide analysis was performed using Progenesis QI for Proteomics (version 4.2), which identified 125 sequences. Of these, 51 sequences were highly confident (scores 4-7) and were frequently reported. Based on the normalised abundance and scores from the LC-MS-MS report, 99.99% of the protein structure was successfully mapped, including regions critical to catalytic activity (\u003cstrong\u003eFig. 2), (Table S3).\u0026nbsp;\u003c/strong\u003eBased on existing literature, five specific epitopes known to contribute to the structural dynamics and catalytic activity of TEM-1 \u0026beta;-lactamase were selected as templates for further nano-MIP synthesis as shown in \u003cstrong\u003eTable 1\u003c/strong\u003e.\u003csup\u003e14-18\u003c/sup\u003e This table details the specific peptide sequences chosen as templates for the synthesis of nano-MIPs. To enable their immobilisation on glass beads for the solid-phase synthesis of nano-MIPs, a terminal cysteine was added to each peptide sequence. This allowed for a stable reaction with the iodoalkyl ends on the IPTMS-silanised glass bead surface.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e(Table 1). Epitopes selected as templates for nano-MIPs synthesis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eResidues \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Template \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSequence\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eDomain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eNotes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCQQLIDWMEADK\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; EP-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; 203 - 213\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eParticipate in the hinge region \u0026alpha; 11 \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAllosteric site\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCLNEAIPNDERDTT\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; EP-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; 166-178\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026Omega; \u0026ndash; loop \u0026amp; Glu 166 - Asn 172\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCTTDREDNPIAENL\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; EP-3\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; 178-166\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026Omega; \u0026ndash; loop \u0026amp; Glu 166 - Asn 172\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eScramble\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCRIHYSQNDLVEYSPVTE\u0026nbsp; \u0026nbsp;EP-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;92-108\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026alpha; 11 and \u0026alpha; 12 -Tyrosine 105\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCMEADKVAGPLLRS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;EP-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp; 209-221\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026alpha; 11 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAllosteric site\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e3.2.\u0026nbsp;Characterisation of synthesised nano-MIPs\u003c/h2\u003e\n\u003cp\u003eBased on the mapped peptides, nano-MIPs were successfully synthesised. The particle sizes of these nano-MIPs, as measured by DLS, ranged from 169 to 242 nm (\u003cstrong\u003eTable S6, Fig.\u003c/strong\u003e \u003cstrong\u003eS9\u003c/strong\u003e). Electron microscopy images from both TEM and SEM further confirmed these DLS measurements, with particle diameters and standard deviations showing high consistency. In some TEM images, nano-MIPs appeared as clustered semi-spherical particles (\u003cstrong\u003eFig.\u003c/strong\u003e \u003cstrong\u003eS10\u003c/strong\u003e), which could be a result of particle aggregation caused by drying. Most of TEM and SEM images revealed the nano-MIPs as discrete, single spherical particles. The final concentration of the nano-MIPs was determined by gravimetric analysis after lyophilisation and by a spectroscopic method using UV absorbance, as detailed in \u003cstrong\u003eFig.\u003c/strong\u003e \u003cstrong\u003eS11\u003c/strong\u003e.\u003c/p\u003e\n\u003ch2\u003e3.3.\u0026nbsp; Analysis of binding kinetics using Octet\u0026reg; BLI\u003c/h2\u003e\n\u003cp\u003eThe optimal assay condition for nano-MIP binding with TEM-1 was at pH 5.0 where better association signal was observed (\u003cstrong\u003eFig. S12)\u003c/strong\u003e. \u0026nbsp;All the synthesised nano-MIPs showed significant affinity and quick saturation (500-600) seconds with TEM-1 \u0026beta;-lactamase (\u003cstrong\u003eTable S4)\u003c/strong\u003e. The association curves showed clear binding signals between nano-MIPs and the enzyme (\u003cstrong\u003eFig.\u003c/strong\u003e \u003cstrong\u003eS12\u003c/strong\u003e), and the affinity of nano-MIPs prepared by imprinting of the mapped epitopes was higher in comparison with nano-MIP@TEM-1, this result supports the efficiency of epitope mapping and epitope imprinting. Furthermore, K\u003csub\u003eD\u003c/sub\u003e values of all synthesised nano-MIPs were lower than NIP (K\u003csub\u003eD\u003c/sub\u003e = 28 nM). It is powerful evidence that confirms the selectivity of the synthesised nano-MIPs. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e3.4.\u0026nbsp;Assessment of enzyme activity and stability of TEM-1 \u0026beta;-lactamase\u003c/h2\u003e\n\u003cp\u003eThe enzyme showed clear increase in the UV absorbance of 40 \u0026micro;M nitrocefin over time at 490 nm. Higher concentrations of TEM-1 showed very fast colour change and absorbance increased very quickly. This confirms the enzyme activity towards the substrate. With lower concentrations, the colour changed gradually; therefore, 2 nM of the enzyme was considered for the assay with nano-MIPs\u0026nbsp;\u003cstrong\u003e(Fig.\u003c/strong\u003e \u003cstrong\u003eS13)\u003c/strong\u003e. Also, the enzyme remained stable and showed similar absorbance curves at the assay conditions, including room temperature and 37 \u003csup\u003eo\u003c/sup\u003eC\u003cstrong\u003e\u0026nbsp;(Fig.\u003c/strong\u003e \u003cstrong\u003eS14)\u003c/strong\u003e. \u0026nbsp;This result is in agreement with Grigorenko et al.\u003csup\u003e39\u003c/sup\u003e, who demonstrated \u0026nbsp;TEM-1 activity at the broad temperature range (41 \u0026ndash; 57) \u0026deg;C.\u003c/p\u003e\n\u003ch2\u003e3.5.\u0026nbsp;Inhibition of TEM-1 activity using nano-MIPs\u003c/h2\u003e\n\u003cp\u003eSignificant reductions in enzyme activity were observed in the presence of nano-MIPs (\u003cstrong\u003eFig. S15\u003c/strong\u003e), indicating enzyme inhibition. While TEM-1 \u0026beta;-lactamase is known for its rigid and conserved structural architecture, its catalytic activity can be significantly altered when a ligand binds to a specific region, potentially blocking the active site. Our results demonstrate that nano-MIPs, synthesised using key epitopes (EP-1 and EP-5 from the allosteric site, EP-2 from the \u0026Omega;-loop, and EP-4 from the \u0026alpha;3-\u0026alpha;4 region, including Tyr105), effectively inhibit the enzyme.\u0026nbsp;This inhibition is consistent with the hypothesis that ligand interactions with the allosteric site (\u0026alpha;11-\u0026alpha;12) can induce dynamic conformational changes in surrounding loops, such as the \u0026Omega;-loop (residues 172-179). The \u0026Omega;-loop plays a crucial role in the catalytic hydrolysis of the \u0026beta;-lactam ring; its fluctuation or rotation, initiated from the allosteric site, can suppress its catalytic enhancement role.\u003csup\u003e40-42\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eQuantitatively, at 20 \u0026deg;C, nano-MIP@TEM-1, nano-MIP@EP-1, and nano-MIP@EP-2 exhibited potent inhibition, exceeding 90% of enzyme activity reduction at concentrations ranging from 1 to 22 pM. Nano-MIP@EP-3, nano-MIP@EP-4, and nano-MIP@EP-5 also showed substantial inhibition, achieving 85%, 79%, and 47% respectively. Notably, at 37\u0026deg;C, all synthesised nano-MIPs demonstrated even higher efficacy, inhibiting more than 93% of the enzyme activity (\u003cstrong\u003eTable 2\u003c/strong\u003e).\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eTable 2. Enzyme inhibition percentage (\u0026gt;90%) by nano-MIPs.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e20C\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e\u0026omicron;\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e37C\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e\u0026omicron;\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003eNano-MIP@ TEM-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e% inhibition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e94 \u0026plusmn;3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e93 \u0026plusmn; 4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eConcentration, pM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e11.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003eNano-MIP@EP-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e% inhibition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e97 \u0026plusmn; 2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e94 \u0026plusmn; 4.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eConcentration, pM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e22.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.44\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003eNano-MIP@EP-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e% inhibition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e99 \u0026plusmn; 1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e99 \u0026plusmn; 0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eConcentration, pM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003eNano-MIP@EP-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e% inhibition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e85 \u0026plusmn; 3.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e99 \u0026plusmn; 0.98\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eConcentration, pM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e45.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e92.34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003eNano-MIP@EP-4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e% inhibition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e79 \u0026plusmn; 2.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e93.57 \u0026plusmn; 0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eConcentration, pM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e184\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e369\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003eNano-MIP@EP-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e% inhibition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e47.6 \u0026plusmn; 7.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e93 \u0026plusmn; 1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eConcentration, pM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e184\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2954\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003eNIP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e% inhibition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eConcentration, pM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe improved efficiency of nano-MIPs against enzyme activity at 37\u0026deg;C can be attributed to higher thermal energy, which enhances both the motion of nano-MIPs and the enzyme, thereby increasing binding by improving the orientational susceptibility of nano-MIP binding sites and imprinted regions. Interestingly, some nano-MIPs demonstrated enzyme inhibition even at low nanoparticles concentrations. Higher concentrations of nano-MIPs in general lead to a stronger inhibitory effect, (\u003cstrong\u003eFig. S16,\u003c/strong\u003e \u003cstrong\u003eFig. S17\u003c/strong\u003e), similar to other antibacterial\u0026nbsp;\u0026beta;-lactamases\u0026nbsp;inhibitors.\u003csup\u003e43-45\u003c/sup\u003e Some deviation from linearity of the response might be explained by aggregation of nanoparticles at room temperature.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNano-MIPs at 37\u0026deg;C showed a clear inhibition dose response across a wide concentration range. The evidence of imprinting effect can be seen from the BLI and enzyme inhibition results obtained for nano-MIP@EP-3. This nano-MIP was synthesised by imprinting EP-3, a scrambled version of EP-2 (with a reversed amino acid sequence). The affinity of nano-MIP@EP-3 (K\u003csub\u003eD\u003c/sub\u003e = 0.18 nM) to enzyme was 30 times lower than that of nano-MIP@EP-2 (K\u003csub\u003eD\u003c/sub\u003e = 0.006 nM). These findings are corroborated by the enzyme inhibition data, where nano-MIP@EP-2 demonstrated a superior inhibitory effect compared to nano-MIP@EP-3.\u003c/p\u003e\n\u003cp\u003eThe MIC results demonstrated that 6.3 \u0026micro;g\u0026times;mL\u003csup\u003e-1\u003c/sup\u003e of ampicillin was the minimum concentration required to suppress \u003cem\u003eE. coli\u003c/em\u003e pET15b growth. The ampicillin MIC assay in CSpET15b revealed clear evidence of AMR: the wild-type (WT) control grew in all CSpET15b wells, whereas it was suppressed by ampicillin in LB medium, and in CSWT at 6.3 \u0026micro;g.mL\u003csup\u003e-1\u003c/sup\u003e (\u003cstrong\u003eFig. S18\u003c/strong\u003e). The Resazurin dye colour change confirmed that WT cells maintained similar viability levels in both WT cell culture and CSWT. Crucially, WT cells continued to grow at all ampicillin concentrations when CSpET15b was present, unequivocally demonstrating the existence of TEM-1 \u0026beta;-lactamase within CSpET15b and its impact on AMR. This finding strongly suggests that TEM-1 \u0026beta;-lactamase is released into the external milieu from bacteria (\u003cstrong\u003eFig. S18\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe results of the disk diffusion filter test further confirmed the ampicillin resistance conferred by the \u003cem\u003eE. coli\u003c/em\u003e pET15b culture supernatant (CSpET15b). As shown in \u003cstrong\u003eFig. S20\u003c/strong\u003e, the growth of ampicillin-sensitive wild-type (\u003cem\u003eE. coli\u003c/em\u003e WT) bacteria around the filter disk loaded with CSpET15b demonstrated that the ampicillin in the agar was degraded by the secreted TEM-1 \u0026beta;-lactamase enzyme. This was in contrast to the control, where the CSWT (which lacks the enzyme) failed to degrade the ampicillin, which inhibited the growth of the WT strain.\u0026nbsp;The antibiotic resistance of the culture supernatant was shown to be concentration-dependent, as reduction in the concentration of the CSpET15b led to a decrease in its ability to protect the WT strain from ampicillin (\u003cstrong\u003eFig.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eS19\u003c/strong\u003e). However, an interesting observation was made when WT strains were exposed to different concentrations of CSpET15b. While WT bacteria exposed to diluted CSpET15b (10%, 1%, 0.1%, and 0.05%) did not grow on ampicillin-agar plates, the WT bacteria exposed to 100% CSpET15b did exhibit resistance. This suggests that the high concentration of TEM-1 \u0026beta;-lactamase in the undiluted supernatant was sufficient to degrade a large amount of ampicillin. Alternatively, this result could be attributed to the transfer of the pET15b plasmid from the culture supernatant to the WT \u003cem\u003eE. coli\u003c/em\u003e cells, a process known as horizontal gene transfer (\u003cstrong\u003eFig. S21\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese findings provide compelling evidence for the release of TEM-1 \u0026beta;-lactamase into the extracellular environment, a phenomenon that is not yet fully verified for all Gram-negative bacteria. While most research suggests that \u0026beta;-lactamases are confined to the periplasmic space or are membrane-bound, some studies, such as that by Schaar et al.\u003csup\u003e19\u003c/sup\u003e, have shown that Gram-negative bacteria like \u003cem\u003eMoraxella catarrhalis\u003c/em\u003e can secrete these enzymes packaged within outer membrane vesicles (OMVs).\u003csup\u003e19\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e46\u003c/sup\u003e \u0026nbsp;This mechanism allows them to inactivate antibiotics and promote the survival of other bacteria. To some extent, the results of this experiment offer new perspectives on the development of antibiotic resistance and highlight the potential for extracellular TEM-1 \u0026beta;-lactamase to play a role in this process.\u003c/p\u003e\n\u003cp\u003eThe activity of the synthesized nano-MIPs was evaluated in both, presence and absence of bacterial cells. When incubated with live \u003cem\u003eE. coli\u003c/em\u003e pET15b cell cultures, the nano-MIPs were unable to suppress antibiotic resistance (\u003cstrong\u003eFig. 3\u003c/strong\u003e). This lack of effect is probably due to the inability of the nano-MIPs to diffuse through the bacterial outer membrane and into the periplasmic space where the TEM-1 \u0026beta;-lactamase is located. Alternatively, the high concentration of the enzyme in the medium may have saturated the limited binding capacity of the nano-MIPs. However, in a supernatant the nano-MIPs demonstrated a clear inhibitory effect. After an overnight incubation with the culture supernatant (CSpET15b), both Nano-MIP@TEM-1 and Nano-MIP@EP-2 were able to reduce the ampicillin resistance of the solution. The minimum inhibitory concentration (MIC) for ampicillin was lowered from 31 \u0026micro;g\u0026times;mL\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eto 15 \u0026micro;g\u0026times;mL\u003csup\u003e-1\u003c/sup\u003e (\u003cstrong\u003eFig. 4\u003c/strong\u003e), demonstrating the ability of the nano-MIPs to effectively suppress the TEM-1 \u0026beta;-lactamase enzyme activity in the supernatant.\u003c/p\u003e"},{"header":"4.\tConclusion","content":"\u003cp\u003eThe increasing capacity of bacteria to deactivate antibiotics poses a major threat to public health and the economy. In this work, we explored the use of nano-MIPs as synthetic alternatives to biological receptors for targeting the TEM-1 \u0026beta;-lactamase enzyme, a key mediator of antibiotic resistance. A novel snapshot imprinting method was used to identify effective binding domains for nano-MIPs on the protein structure. Crucially, this included the identification of domains known to be connected to the enzyme\u0026apos;s active site (Ser70), such as the \u0026alpha;3-\u0026alpha;4 loop, the \u0026Omega;-loop, and the allosteric site. Five of these mapped epitopes were selected as templates for synthesising nano-MIPs using a solid-phase protocol. The resulting nano-MIPs were spherical, with particle sizes ranging from 169 to 242 nm.\u003c/p\u003e\n\u003cp\u003eThe binding kinetics of these nano-MIPs were evaluated using Octet\u0026reg; Biolayer Interferometry (BLI). The data showed that the nano-MIPs possessed excellent affinity and selectivity for TEM-1, with picomolar-scale affinity constants (K\u003csub\u003eD\u003c/sub\u003e). Furthermore, a functional assay demonstrated that the nano-MIPs were able to inhibit more than 93\u0026ndash;99% of the enzyme\u0026apos;s activity. We hypothesise that this inhibition is a result of the nano-MIPs binding to key domains on the enzyme, which disturbs its conformational dynamics and prevents the antibiotic substrate from accessing the active site.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo investigate the phenomenon of TEM-1 \u0026beta;-lactamase release, we studied the antibiotic resistance of \u003cem\u003eE. coli\u003c/em\u003e pET15b in both the cell culture and its supernatant. The results indicated that the culture supernatant conferred an antibiotic resistance level equivalent to that of the live cell culture. This finding was further supported by a disk diffusion test and a titration of the supernatant\u0026apos;s antibiotic resistance, providing evidence that TEM-1 \u0026beta;-lactamase is released beyond the bacterial outer membrane. When tested against live bacterial cultures, the nano-MIPs were ineffective at suppressing antibiotic resistance. This is likely due to their inability to diffuse into the periplasmic space to interact with \u0026nbsp;the enzyme. However, in the cell-free culture supernatant, the nano-MIPs successfully suppressed AMR, reducing the minimum inhibitory concentration (MIC) of ampicillin from 31\u0026nbsp;\u0026micro;g\u0026times;mL\u003csup\u003e-1\u003c/sup\u003e to 15\u0026nbsp;\u0026micro;g\u0026times;mL\u003csup\u003e-1\u003c/sup\u003e. Future work could focus on designing MIPs to target well-documented outer membrane proteins, such as porins, to explore new strategies for killing bacteria by deactivating or blocking essential cellular functions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eA. Valsamatzi-Panagiotou, M. Traykovska and R. Penchovsky, \u003cem\u003eDrug Discovery Targeting Drug-Resistant Bacteria\u003c/em\u003e, 2020, 9-37.\u003c/li\u003e\n\u003cli\u003eL. M. Lima, B. N. M. da Silva, G. Barbosa and E. J. 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Lin, \u003cem\u003eFrontiers in microbiology\u003c/em\u003e, 2013, \u003cstrong\u003e4\u003c/strong\u003e, 128.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Leicester","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Molecularly Imprinted Polymer nanoparticles (nano-MIPs), Antimicrobial Resistance, TEM-1 β-lactamase, Snapshot Imprinting, Enzyme Inhibition.","lastPublishedDoi":"10.21203/rs.3.rs-8721892/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8721892/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAntimicrobial resistance (AMR) is a major global health threat, as classified by the World Health Organisation (WHO), and is driven by the ability of microorganisms to deactivate drugs. This research aims to address AMR by employing molecularly imprinted polymer nanoparticles (nano-MIPs) capable of inhibiting TEM-1 β-lactamase enzyme, a key contributor to bacterial drug resistance. We used a snapshot imprinting technique to map the epitopes of the TEM-1 enzyme. This mapping identified five crucial epitopes, which were then used to synthesize the nano-MIPs via a solid-phase protocol. The resulting nano-MIPs with 169-242 nm diameter, demonstrated exceptional affinity and selectivity for TEM-1, with dissociation constants (K\u003csub\u003eD\u003c/sub\u003e) as low as 0.006–0.35 nM. In vitro assays confirmed the effectiveness of the nano-MIPs in inhibiting 93% of the TEM-1 activity. We also investigated antibiotic resistance in both E. coli pET15b cell cultures and the culture supernatant, where TEM-1 is released. Initial tests revealed significant resistance to ampicillin in the culture supernatant, which nano-MIPs successfully mitigated. The nanoparticles were able to reduce the effective dose of the antibiotic from 31 µg×mL\u003csup\u003e-1\u003c/sup\u003e to 15 µg×mL\u003csup\u003e-1\u003c/sup\u003e. The high efficiency of nano-MIPs suggests that this approach could be broadly applied to target other critical biomarkers involved in bacterial survival, offering a promising new strategy to combat AMR.\u003c/p\u003e","manuscriptTitle":"Design of Molecularly Imprinted Polymer Nanoparticles Capable of Suppressing TEM-1 β-Lactamase Activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-04 09:10:31","doi":"10.21203/rs.3.rs-8721892/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"36f4a4d8-b652-42df-8356-4ff319c22a34","owner":[],"postedDate":"February 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61906051,"name":"Polymer Science"},{"id":61906052,"name":"Nanoscience"},{"id":61906053,"name":"Analytical Biochemistry"},{"id":61906054,"name":"Chemical Biology"}],"tags":[],"updatedAt":"2026-02-04T09:10:35+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-04 09:10:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8721892","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8721892","identity":"rs-8721892","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}