Ultra-Sensitive Hydrogen Sulfide Detection via Hybrid Small-Molecule Nano-arrays

Ultra-Sensitive Hydrogen Sulfide Detection via Hybrid Small-Molecule Nano-arrays | 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 Article Ultra-Sensitive Hydrogen Sulfide Detection via Hybrid Small-Molecule Nano-arrays Zhenda Lu, Xing xing, Luyan Wu, Yuchen Zhang, Jiahao Pan, Yusuke Ishigaki, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4455040/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Dec, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Early disease diagnosis hinges on the sensitive detection of signaling molecules. Among these, hydrogen sulfide (H 2 S) has emerged as a critical player in cardiovascular and nervous system signaling. On-chip immunoassays, particularly nanoarray-based interfacial detection, offer promising avenues for ultra-sensitive analysis due to their confined reaction volumes and precise signal localization. Beyond the DNA or protein biomolecules array, this work presents a promising hybrid small molecule nano-array for H 2 S detection, using the power of dual molecules: a dye for fluorescence emission and a quencher with specific H 2 S reactivity. Upon H 2 S interaction, the quenched fluorescence reignites, creating an easily detectable array of bright spots. The molecule nano-array sensor showed exceptional responses to H 2 S over 8 magnitudes of dynamic range from 1 fM to 0.1 μM, with a remarkable detection limit of 1 fM, just using a 10 μL solution. This new H 2 S detection method has the potential to significantly improve bioassay platforms, and the hybrid small-molecule nano-arrays we developed could be a valuable tool for advancing signaling molecule detection. Physical sciences/Nanoscience and technology/Nanobiotechnology/Nanofabrication and nanopatterning Physical sciences/Nanoscience and technology/Nanobiotechnology/Biosensors Physical sciences/Nanoscience and technology/Nanobiotechnology/Applications of AFM molecule nano-arrays ultra-sensitive detection hydrogen sulfide fluorescence probe nanoxerography Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Hydrogen sulfide (H 2 S) has been recognized as a signaling molecule in the cardiovascular and nervous systems, making its accurate detection vital for diagnosing and managing related diseases. 1 – 5 While baseline H 2 S levels hover around micromolar, detecting even lower concentrations holds immense promise, offering a previous window for the early-stage illness diagnosis and intervention before symptoms manifest. 6 – 17 To meet the demand, a recent study introduced F1 2+ , a bis(pentafluorophenyl)-substituted organic π-electron structure. 18 , 19 F1 2+ boasts impressive characteristics: rapid reaction kinetics to H 2 S with excellent specificity, minimizing false positives. 18 Utilizing this remarkable molecule, researchers achieved an impressive detection limit of 0.1 µM endogenous H 2 S in vitro, showing its potential for sensitive biomedical applications. 18 However, pushing the boundaries of detection even further demands innovative approaches. On the other hand, micro-arrays have long been an invaluable tool for rapid, high-throughput, and sensitive analysis. 20 – 27 Their well-arranged molecular spots offer enhanced accuracy and quantitative precision, resulting in a clear contrast to the random molecules. Advancements in micro-nano fabrication have yielded dense micro-arrays, exceeding 1000 spots per square millimeter. 22 But nano-arrays, offer even higher density and miniaturized reaction spaces. 26 , 28 , 29 Imagine hundreds of reaction tests can occur in the area of a single conventional microarray spot, enabling the performance of ultra-sensitive detection, rapid analysis, and small sample volumes. However, precisely positioning and integrating multiple molecules on the nanoscale remains a formidable challenge. 30 – 34 While existing nano-arrays focus on larger biomolecules like DNA and proteins, the small-molecule nano-arrays, are rarely reported. 35 – 41 This is, in part, due to the inherent nature of small molecules – they are difficult to manipulate, lack strong bonds with the substrate, and are hard to detect. Here, we present a molecule nano-array fabrication method based on a modified nano-xerography technique, 42 – 47 precisely co-assembling Rhodamine B (RB) dye and F1 2+ molecules into nano-arrays, with a spot size of ~ 100 nm and a spot-to-spot pitch of 3–5 µm. The spot, as a nanoreactor, is capable of capturing trace amounts of H 2 S from test solutions, decomposing the quencher F1 2+ molecules, and then reigniting the RB fluorescence. This approach at least has several key advantages: 1) ultra-sensitive and rapid detection: nanoscale reaction areas make the array quantitatively responsive to trace amounts of H 2 S, reaching detection limits of 1 fM. Also, the small size of the spots allows for quick and real-time detection, which is crucial in biological diagnostics. 2) broad-range detection: different data processing methods (NUMB or INTEN) depending on the analyte concentration can be applied, making a detection range of H 2 S that spans over 8 orders of magnitude, from 1 fM to 0.1 µM. 3) specific detection: taking advantage of the chemical affinity system, the high H 2 S specificity of F1 2+ ensures sensitive and reliable detection. Combining the power of F1 2+ and molecule nano-arrays, this innovative approach paves a new way of ultra-sensitive H 2 S detection. Moreover, this kind of molecule nano-arrays can extend to other molecular fluorescence probe systems, making it universal to develop an innovative analysis method of the small signaling molecules in biological systems. Results and discussion General description of hybrid molecule nano-array. The molecule nano-array sensor, which is schematically shown in Fig. 1 a, comprises a spot of hybrid molecules with each spot diameter of ~ 100 nm (Figure S1 ). Among them, the hybrid molecules consist of fluorescence-quencher molecules (F1 2+ ), dye molecule RB, and cyclodextrin. F1 2+ molecule has wide fluorescence absorption capacity in the visible light range, especially in the 500–650 and 700–900 nm, which covers the excitation and emission wavelength of most dye molecules. RB dye molecule is selected as the fluorescence agent, a common dye molecule with absorption and emission center wavelengths of 530 nm and 590 nm, respectively, that can be quenched by F1 2+ molecule effectively. Cyclodextrin was used as a space barrier to prevent aggregation-caused quenching (ACQ) of dye molecules. The absorption spectra of the separated three molecules and their combination were shown in Figure S2a, which were characterized by spin-coating the corresponding molecules onto a cover glass. F1 2+ can be quickly reduced to an F2 molecule by H 2 S (Fig. 1 b), which has almost no absorption in the visible light range (Figure S2b). Based on this reaction platform, we printed the hybrid molecules with certain proportions into nano-arrays on the substrate by the modified nanoxerography method we developed. 42 , 43 At each spot, the RB fluorescence emitted is quenched by F1 2+ molecules, so that the nano-arrays before reacting with H 2 S showed very weak fluorescence. When F1 2+ is decomposed by H 2 S molecules, the fluorescence of the RB regenerates, which can be easily imaged by a wide-field fluorescence microscope. The fluorescent change of each spot in the nano-array correlates with the H 2 S concentration. We utilize an intensity integral (INTEN) and number counting (NUMB) method to quantify the fluorescence activities on the spots at high and low H 2 S concentrations, respectively (Fig. 1 c). 48 , 49 The INTEN method surveys all the spots in the nanoarray and integrates the fluorescence intensity, which can detect H 2 S concentration from 0.3 pM to 0.1 µM. At low concentrations, only parts of the spots have measurable fluorescence changes (defined as “on”). The NUMB method counts the separated “on” spots, showing a good correlation with the H 2 S concentration in the range of 1 fM to 100 pM, two orders of magnitude lower than that of INTEN method. The detailed configurations of INTEN and NUMB methods are listed in the Experimental section. Preparation of hybrid molecule nano-array. The molecule nano-array was fabricated by the modified nano-xerography method we had reported previously. 42 , 43 The typical procedures are schematically shown in Fig. 2 a. First, a local surface charge array was written on the substrate by controlling a conductive AFM probe to apply high voltage (~ -80V), which can generate − 2.4 V charge potential on the substrate, as proved by the Kelvin probe force microscopy (KPFM) potential maps in Fig. 2 b. Then, F1 2+ , RB, and cyclodextrin molecules were dissolved in water as a dispersed phase to form water-in-oil reverse micelles, with hexane as a continuous phase and AOT as a surfactant. The micelles loaded with F1 2+ , RB, and cyclodextrin were spin-coated on the substrate. During this process, the molecules would be enriched and adsorbed onto the charged array. In the uncharged area, the perfluorinated substrate (CYTOP) could effectively avoid non-specific adsorption due to its ultra-low surface energy. As shown in AFM height map (Fig. 2 c), a 5×5 molecular nano-array with a spot size of ~ 200 nm and a spacing of 5 µm was successfully assembled with less nonspecific adsorption. The micelle dispersion was characterized by dynamic light scattering (DLS) and Zeta potential measurement. The DLS result (Fig. 2 d) demonstrates the average micelle size in solution is around 3 nm, which is consistent with the results previously reported in the literature. 42 , 50 The zeta potential is around − 31.1 ± 11.0 mV, indicating the strong negative charge and the good stability of the micelles. To investigate the steric spacer effect of cyclodextrin (CD) in the hybrid molecular system, we prepared RB micelles and RB/CD mixture micelles (1:1 molar ratio) and assembled them onto charged spots with identical surface potential. As expected, no fluorescence array was observed, attributed to aggregation-caused quenching (ACQ) of RB molecules (Figure S3a). Upon the addition of CD, a robust fluorescence nano-array emerged (Figure S3b). Subsequently, RB/CD was combined with the quencher molecule F1 2+ to form the final RB/CD/F1 2+ system (1:1:2 molar ratio). The average fluorescence intensity of the spots decreased significantly, reaching only 9% compared to the case without F1 2+ molecules (Figure S3c). Despite efforts to increase F1 2+ concentration, complete elimination of fluorescence proved challenging. This difficulty may arise from RB fluorescence being emitted in multiple directions, making it hard to complete quenching. Due to its promising characteristics, this RB/cyclodextrin/F1 2+ hybrid molecular system (1:1:2 molar ratio) was chosen for subsequent micro-area fluorescence detection (Fig. 2 e). Typical PL images of 5×5 nano-array before and after reacting with H 2 S solution are shown in Fig. 2 f. After the reaction, the fluorescence intensity of each hybrid molecule spot ranges from 300 to over 15000 arbitrary units (a.u.), compared to less than 290 a.u. of the negative controls (incubated with water). The fluorescence intensity on each spot is enhanced more than 50 times. These results clearly demonstrate the enormous potential of the nano-array in ultra-sensitive small molecule detection. Quantitatively H 2 S detection at high concentrations using INTEN method. We quantificationally investigated the luminescence response of the hybrid molecule nano-array toward H 2 S. 10 µL of NaHS aqueous solution ranging from 0.1 fM to 1 µM were tested by the hybrid molecule nano-arrays (5×5 square spots). Figure 3 a shows the fluorescence images of the nano-array after sulfide assay from 10 pM to 1 µM. The images related to the other concentrations are shown in Figure S4. It is clear that the fluorescence intensity on each spot have a positive correlation with the concentration. All the spots are illuminated when the concentration is above 100 pM, on the contrary, only parts of spots are detectable in the images below 10 pM. To quantitatively analyze the correlation, we read out the fluorescence intensity of each spot. (Detailed information are shown in the Method section, and Figure S5-S6). If the intensity is larger than that before NaHS assay, we considered it an illuminated spot (“on” spot). The intensity sum of all the “on” spot versus corresponding concentration were plotted (Fig. 3 b), noted as INTEN method. The plot is fitted with the well-established four-parameter logistic function curve (4-PL fitting), showing a good dynamic detection range over 5 magnitudes, from 300 fM to 1 µM. The linear range after logit transformation was between 100 pM and 100 nM, in which the spots are all reignites. Quantitatively H 2 S detection at low concentrations using NUMB method. As the NaHS concentration was below 10 pM, the sum of fluorescence intensity of the “on” spot is relatively low to quantitative analysis, and parts of the spots hold the “off” state (Figure S4). This is because not all the spots have the chance to react with hydrogen sulfide in several minutes at very low concentration. In view of this, we counted “on” spot numbers, and correlated them with the NaHS concentrations, noted as NUMB method. To get more reliable results at low concentration of NaHS, we increased the spot number to 400 by designing a 20×20 array pattern (Fig. 4 ). NaHS solutions with concentrations from 0.1 fM to 1 nM were prepared. Figure 4 a shows the fluorescence images corresponding to the concentration from 1fM to 100 pM, and all the “on” spots marked with circles. The 20×20 molecule nano-array showed the NaHS detection limit of around 70 fM using INTEN method (Fig. 4 b). In comparison, the detection limit is about 300 fM with the same method when utilizing the 5×5 nanoarray, indicating more spots can lead to a lower detection limit. The images were further characterized by NUMB method to extend the NaHS detection limit to an even lower concentration (Fig. 4 b). After 4-PL fitting, a good correlation between the number of “on” spots and the concentration of NaHS from 1 fM to 100 pM is demonstrated with the R 2 value of 0.99 (Table S1 ), indicating exceptional dynamic detection range. The detection limit has been reduced by about 2 orders of magnitude, compared to the INTEN method. Combined with the results from INTEN method at high NaHS concentrations and NUMB method at low NaHS concentrations, this hybrid molecule nano-array platform has achieved the detection dynamic range from 1 fM to 0.1 µM, about 8 orders of magnitudes of response. This is a significant improvement in detection range and detection limit compared to the existing method for signaling small molecules. Conclusion We have developed a hybrid molecule nano-array sensor and implemented it for the highly sensitive detection of hydrogen sulfide. The nano-array features a small reaction volume, enabling a specific response from the F1 2+ molecule designed for H 2 S detection. With its high-sensitivity turn-on fluorescence detection mechanism, this nano-array achieves an exceptional detection limit of 1 fM using just a 10 µL solution. Furthermore, it exhibits a broad detection dynamic range spanning 8 orders of magnitudes. The outstanding performance of this nano-array is attributed to the robust nanoxerography technique and the use of well-developed molecular fluorescence probes. Notably, the versatility of this molecule nano-array extends beyond H 2 S detection, making it universally applicable to detect other signaling small molecules. This characteristic demonstrates its great potential in the bio-detection applications. Methods Chemicals and characterization. Sodium bis(2-ethylhexyl) sulfosuccinate (AOT, MACKLIIN, 96%), Rhodamine (RB, Aladdin, > 95.0% (HPLC)), β-Cyclodextrin (CD, Aladdin, 98%), Isooctane (Aladdin, 99%), cyclohexane (Shanghai Lingfeng Chemical Reagent Co. Ltd, ≥ 99.7%, AR), DMSO (Sinopharm Chemical Reagent Co., Ltd, 99.0%), CYTOP (ASAHI GLASS COMPANY, Japan), F1 2+ (Deju Ye group, Nanjing University). All chemicals were used as received without further purification. Preparation of reverse micelle solutions. 50 The dye and F1 2+ stock solutions were prepared by dissolving dye molecule (RB or 1:1 RB/CD) in water and F1 2+ powders in water solution with a very small amount of DMSO, respectively. Then, RB/F1 2+ stock solution (molar ratio of 1:2) and RB/F1 2+ /CD stock solution (molar ratio of 1:2:1) were prepared with overall molecule concentration of 5.0 mM. The reverse micelle solutions were prepared by injecting 10 µL aqueous stock molecular solution into 82.6 µL AOT/isooctane solution with ultrasonication until forming clear and transparent solution. The concentration of AOT/isooctane was fixed at 1.12 M and thus the ω value of micelle solutions was kept at 6.0. Dynamic light scattering (DLS) and zeta potential measurements were performed on a 90 Plus/BI-MAS equipment (Brookhaven, USA). Fabrication of molecule nano-array. 42 , 43 The molecule nano-array was fabricated by modifying electric field-assisted surface sorption nano-printing (EFASP) method which consists of two stpes: charge array writing on a fluorine polymer electret (CYTOP) and micelle assembly on the charged array. In a typical process, fluorine polymer CYTOP was spin-coated on Si substrate which was pre-marked by lithography and baked to remove extra solvent for forming a 110 nm thick film (electret). Then, by applying high voltage (-40V to -90V) on a conductive AFM tip and writing charge on the electret film with pre-designed array pattern based on AFM scanning system, surface charge arrays (-0.15 to -3.0 V) were generated. At the same time, local surface modification occurs at the charged position, Kevin probe scanning (KPFM) measurements were determined whether the charge array was written successfully. All the charge writing process was performed in the air under ambient conditions (room temperature, relative humidity = 20–40%). The charge writing and KPFM scanning were performed on a NT-MDT AFM instrument. After that, water-in-oil reverse micelle solution was dropped onto the charged area and tilted it after immersed 30s. Micelles containing hybrid molecules were captured to the charged arrays locally. The morphology after micelle assemblies is characterized by the AFM tapping mode scanning. Detection of hydrogen sulfide. A thin layer of CTYOP (10–30 nm) was spun on the assembled hybrid molecule nano-array to protect it from being rinsed off during the H 2 S detection. The CYTOP concentration, spin speed, spin time, and acceleration were set as 2%, 7000 rpm, 1 min and 2000 r/min 2 . There is no change in fluorescence image including fluorescence intensity and spots position before and after CYTOP covering. NaHS aqueous solution with concentrations were first prepared ranging from 0.1 fM to 1 µM and used immediately after preparation (less than 5 minutes). During the detection, NaHS aqueous solutions (10 µL) with different concentrations were dropped onto different molecular nano-arrays and blown away by N 2 stream after 2 min. Finally, The nano-arrays were placed on a home-made wide-field inverted fluorescence microscope and their fluorescence photos were taken every 20 seconds. The fluorescence microscope was set up by Olympus inverted fluorescence microscope with PI spectrometer and EMCCD. The 532 continuous-wave laser is fiber-coupled and focused on the focal plane behind the objective lens through a long focal length lens. After passing through the 40× objective lens, a spot of light about 120 microns in diameter focused on the sample. The emitted fluorescence is collected by an EMCCD after passing through the color filter with exposure time of 1s. The power of the excited light is fixed at 30 µW. Data Analysis. Image analysis was performed in MATLAB. Each original fluorescence image was calculated by subtracting the mean background value (M bg ) and all the background (light intensity below 20 after subtracting M bg ) was set to 0. We take these results as effective fluorescence images. Then we divided each effective fluorescence image into small images so that each small image contains one spot (Figure S5 and S7). We calculated the sum of fluorescence intensity (I sum ) of each spot. If the I sum of a spot is larger than the average I sum of spots before NaHS reaction, we considered it a illuminated spot (“on” spot). The average fluorescence intensity or the number of “on” spots were used to establish functional relationship with the concentration of NaHS, noted as INTEN and NUMB method, respectively. Calibration curves were fit using a four-parameter logistic (4-PL) fit in Origin software. The values of fitting parameters are listed in Table S1 . A linear regression was applied to the steepest part of the calibration curve. The background level is defined as the negative control (identical procedures using water instead of NaHS solutions) plusing three times of the standard deviation. The intersection of the regression line and the background level defined the LOD. Notes The authors declare no competing financial interest. 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J., Hlavacek, A., Skladal, P. & Gorris, H. H. Single molecule upconversion-linked immunosorbent assay with extended dynamic range for the sensitive detection of diagnostic biomarkers. Anal. Chem. 89 , 11825-11830 (2017). Zang, F. et al. Ultrasensitive ebola virus antigen sensing via 3D nanoantenna arrays. Adv. Mater. 31 , 1902331 (2019). Wu, M. L., Chen, D. H. & Huang, T. C. Synthesis of Au_Pd Bimetallic Nanoparticles in Reverse Micelles. Langmuir 17 , 3877-3883 (2001). Additional Declarations There is NO Competing Interest. Supplementary Files SupportingInformation0521.docx Cite Share Download PDF Status: Published Journal Publication published 30 Dec, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4455040","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":311350449,"identity":"0cf3663e-f978-4e35-afd6-9726df36c4aa","order_by":0,"name":"Zhenda Lu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYBACPgYGNiDFzMDPwJAAZhAEbDAtkg0kazE4AOYTo0Ui/dmDjzus5Y1vNzyTYKiwTmxgP3uAgJaEdMOZZ9INt905kCbBcCY9sYEnL4GQlmPSvG2HGbfdSEiTYGw7nNggwWNAQEtim/TftsP2m2eAtPwjSksymzTI8A0SIC0NxGjhecYm2duWnjzjRkKyRcKxdOM2nhz8WvjZ059J/Gyztu2fkZN440ONtWw/+xn8WpAATwI4MtmIVQ8E7AdIUDwKRsEoGAUjCQAALOA/ttwhZdMAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-9616-8814","institution":"Nanjing University","correspondingAuthor":true,"prefix":"","firstName":"Zhenda","middleName":"","lastName":"Lu","suffix":""},{"id":311350450,"identity":"97ce6fd8-f53f-42c1-855e-c8ee023608f4","order_by":1,"name":"Xing xing","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Xing","middleName":"","lastName":"xing","suffix":""},{"id":311350451,"identity":"79bec445-c04d-4a9a-bd76-d39b9c1242a1","order_by":2,"name":"Luyan Wu","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Luyan","middleName":"","lastName":"Wu","suffix":""},{"id":311350452,"identity":"601ad278-8e9f-4dff-8a6d-56e5566eb4da","order_by":3,"name":"Yuchen Zhang","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Yuchen","middleName":"","lastName":"Zhang","suffix":""},{"id":311350453,"identity":"0111ffa4-a3ab-4df2-a478-e5b7da76d02f","order_by":4,"name":"Jiahao Pan","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Jiahao","middleName":"","lastName":"Pan","suffix":""},{"id":311350454,"identity":"6a5ed0a2-bc86-4080-a49c-c5891f60a53c","order_by":5,"name":"Yusuke Ishigaki","email":"","orcid":"https://orcid.org/0000-0001-7961-3595","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Yusuke","middleName":"","lastName":"Ishigaki","suffix":""},{"id":311350455,"identity":"441bc8a8-2824-422c-bfdb-6b242cd28869","order_by":6,"name":"Takanori Suzuki","email":"","orcid":"https://orcid.org/0000-0002-1230-2044","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Takanori","middleName":"","lastName":"Suzuki","suffix":""},{"id":311350456,"identity":"1ce56210-93b6-461f-aad7-e8987c13d231","order_by":7,"name":"Deju Ye","email":"","orcid":"https://orcid.org/0000-0002-9887-0914","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Deju","middleName":"","lastName":"Ye","suffix":""},{"id":311350457,"identity":"8a1013bc-29fd-43ad-a247-f33b3d1190cd","order_by":8,"name":"Weihua Zhang","email":"","orcid":"https://orcid.org/0000-0003-1743-5919","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Weihua","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-05-21 13:05:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4455040/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4455040/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-024-55123-y","type":"published","date":"2024-12-30T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57888859,"identity":"02e5b237-99c6-4a2c-835c-ab3873041c01","added_by":"auto","created_at":"2024-06-07 05:48:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":99431,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIllustration of highly sensitive H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eS detection via a hybrid molecule nano-array. \u003c/strong\u003e(a) The design of a hybrid molecule nano-array for H\u003csub\u003e2\u003c/sub\u003eS detection, involving F1\u003csup\u003e2+\u003c/sup\u003e, RB, and cyclodextrin (CD) molecules. During the process of H\u003csub\u003e2\u003c/sub\u003eS detection, the spots within the molecule array transition from the fluorescence \"off\" state to an \"on\" state. By integrating the fluorescence intensity (INTEN method) at high concentrations or counting the number of the “on” spots (NUMB method), the correlation with H\u003csub\u003e2\u003c/sub\u003eS concentration can be established. (b) The chemical equation of F1\u003csup\u003e2+\u003c/sup\u003e to F2 reduced by H\u003csub\u003e2\u003c/sub\u003eS. (c) The typical fluorescent image of the array and the corresponding correlation spots obtained by INTEN and NUMB methods at high H\u003csub\u003e2\u003c/sub\u003eS concentration and low concentration respectively.\u0026nbsp;\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4455040/v1/4de51352e0b84f4325ef8e31.png"},{"id":57888364,"identity":"ae95d9a2-8f10-4bb0-91d4-427727632fc5","added_by":"auto","created_at":"2024-06-07 05:40:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":101011,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Schematic illustration of fabricating hybrid molecule nano-array using modified nanoxerography method, which involves surface charge writing, reversed-micelle preparation, and site-specific assembly by electrical trapping. (b) Electric potential map after writing a 5×5 charge array on the substrate. Scale bar, 5 μm. (c) AFM height map of the hybrid molecule nano-array, with an average height of 50 nm. Scale bar, 5 μm. (d) Dynamic light scattering (DLS) result of reversed-micelles, with the statistic size in dispersion around 3 nm. Inset is a photograph of the micelle dispersion. (e) Scheme of the fluorescence array imaging reader. A homemade inverted widefield microscope is equipped with a fiber-coupled 532 nm continuous-wave laser diode and a sensitive EMCCD camera. (f) Schematic illustration of H\u003csub\u003e2\u003c/sub\u003eS detection, and corresponding PL images of the hybrid molecule array after incubation with water (10 μL) and H\u003csub\u003e2\u003c/sub\u003eS solution (10 μL, 0.1 μM). Scale bar, 5 μm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4455040/v1/fe2a8b9cd8c2547f7fc1e304.png"},{"id":57888369,"identity":"6adef099-71e5-4cb0-aac3-9340cf90a9f7","added_by":"auto","created_at":"2024-06-07 05:40:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":72844,"visible":true,"origin":"","legend":"\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eS detection at high concentration using INTEN method. (a) PL images of serial NaHS concentration in 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, and 1 μM in the 5×5 nano-assay. The images of other concentrations (from 0.1 fM to 1 pM) are shown in Figure S6. Scale bar, 5 μm. (b) The working curve of NaHS tested on the nanoarray using the INTEN method. The black line shows the fitting result by the 4-parameter logistic regression model. The blue dotted line represents the background levels, which are defined as the mean luminescence intensity of negative controls with a three-fold standard deviation. The fitting parameters are listed in Table S1. The blue dash line shows the linear regression after logit transformation of the method.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4455040/v1/3b81dc4577529257b86939f4.png"},{"id":57888366,"identity":"1a3b0483-4cc2-49ca-8435-e04794f58346","added_by":"auto","created_at":"2024-06-07 05:40:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":91932,"visible":true,"origin":"","legend":"\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eS detection at low concentration using \u003cstrong\u003eNUMB\u003c/strong\u003e method. (a) PL images of serial NaHS concentration in 1 fM, 10 fM, 100 fM, 1 pM, 10 pM and 100 pM in the assay of 20×20 molecules nano-array. Scale bar, 9 μm. (b) The fitting results of the NUMB and INTEN methods. The 4-parameter logistic regression model yields an LOD of 1 fM in the \u003cstrong\u003eNUMB\u003c/strong\u003e method and 70 fM in the \u003cstrong\u003eINTEN\u003c/strong\u003e method. The dotted line represents the background levels, which are defined as the mean luminescence intensity of negative controls with a three-fold standard deviation. The fitting parameters are listed in Table S1.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4455040/v1/758bc508f0f4ff23a560776e.png"},{"id":72685877,"identity":"8bb1231b-674f-4ed2-b482-c57cfc9d9b52","added_by":"auto","created_at":"2024-12-31 08:21:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":912066,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4455040/v1/d30b3f00-386b-4611-af12-2c94d9b1a6f5.pdf"},{"id":57888368,"identity":"c21acdec-d323-4531-9ef4-09d52c04c99a","added_by":"auto","created_at":"2024-06-07 05:40:56","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2540773,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupportingInformation0521.docx","url":"https://assets-eu.researchsquare.com/files/rs-4455040/v1/4b25f03ad8df5d3675abe63e.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Ultra-Sensitive Hydrogen Sulfide Detection via Hybrid Small-Molecule Nano-arrays","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS) has been recognized as a signaling molecule in the cardiovascular and nervous systems, making its accurate detection vital for diagnosing and managing related diseases.\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e While baseline H\u003csub\u003e2\u003c/sub\u003eS levels hover around micromolar, detecting even lower concentrations holds immense promise, offering a previous window for the early-stage illness diagnosis and intervention before symptoms manifest.\u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10 CR11 CR12 CR13 CR14 CR15 CR16\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTo meet the demand, a recent study introduced F1\u003csup\u003e2+\u003c/sup\u003e, a bis(pentafluorophenyl)-substituted organic π-electron structure.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e F1\u003csup\u003e2+\u003c/sup\u003e boasts impressive characteristics: rapid reaction kinetics to H\u003csub\u003e2\u003c/sub\u003eS with excellent specificity, minimizing false positives.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Utilizing this remarkable molecule, researchers achieved an impressive detection limit of 0.1 \u0026micro;M endogenous H\u003csub\u003e2\u003c/sub\u003eS in vitro, showing its potential for sensitive biomedical applications.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e However, pushing the boundaries of detection even further demands innovative approaches.\u003c/p\u003e \u003cp\u003eOn the other hand, micro-arrays have long been an invaluable tool for rapid, high-throughput, and sensitive analysis.\u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24 CR25 CR26\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Their well-arranged molecular spots offer enhanced accuracy and quantitative precision, resulting in a clear contrast to the random molecules. Advancements in micro-nano fabrication have yielded dense micro-arrays, exceeding 1000 spots per square millimeter.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e But nano-arrays, offer even higher density and miniaturized reaction spaces.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Imagine hundreds of reaction tests can occur in the area of a single conventional microarray spot, enabling the performance of ultra-sensitive detection, rapid analysis, and small sample volumes.\u003c/p\u003e \u003cp\u003eHowever, precisely positioning and integrating multiple molecules on the nanoscale remains a formidable challenge.\u003csup\u003e\u003cspan additionalcitationids=\"CR31 CR32 CR33\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e While existing nano-arrays focus on larger biomolecules like DNA and proteins, the small-molecule nano-arrays, are rarely reported.\u003csup\u003e\u003cspan additionalcitationids=\"CR36 CR37 CR38 CR39 CR40\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e This is, in part, due to the inherent nature of small molecules \u0026ndash; they are difficult to manipulate, lack strong bonds with the substrate, and are hard to detect.\u003c/p\u003e \u003cp\u003eHere, we present a molecule nano-array fabrication method based on a modified nano-xerography technique,\u003csup\u003e\u003cspan additionalcitationids=\"CR43 CR44 CR45 CR46\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e precisely co-assembling Rhodamine B (RB) dye and F1\u003csup\u003e2+\u003c/sup\u003e molecules into nano-arrays, with a spot size of ~\u0026thinsp;100 nm and a spot-to-spot pitch of 3\u0026ndash;5 \u0026micro;m. The spot, as a nanoreactor, is capable of capturing trace amounts of H\u003csub\u003e2\u003c/sub\u003eS from test solutions, decomposing the quencher F1\u003csup\u003e2+\u003c/sup\u003e molecules, and then reigniting the RB fluorescence. This approach at least has several key advantages: 1) ultra-sensitive and rapid detection: nanoscale reaction areas make the array quantitatively responsive to trace amounts of H\u003csub\u003e2\u003c/sub\u003eS, reaching detection limits of 1 fM. Also, the small size of the spots allows for quick and real-time detection, which is crucial in biological diagnostics. 2) broad-range detection: different data processing methods (NUMB or INTEN) depending on the analyte concentration can be applied, making a detection range of H\u003csub\u003e2\u003c/sub\u003eS that spans over 8 orders of magnitude, from 1 fM to 0.1 \u0026micro;M. 3) specific detection: taking advantage of the chemical affinity system, the high H\u003csub\u003e2\u003c/sub\u003eS specificity of F1\u003csup\u003e2+\u003c/sup\u003e ensures sensitive and reliable detection.\u003c/p\u003e \u003cp\u003eCombining the power of F1\u003csup\u003e2+\u003c/sup\u003e and molecule nano-arrays, this innovative approach paves a new way of ultra-sensitive H\u003csub\u003e2\u003c/sub\u003eS detection. Moreover, this kind of molecule nano-arrays can extend to other molecular fluorescence probe systems, making it universal to develop an innovative analysis method of the small signaling molecules in biological systems.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e \u003cb\u003eGeneral description of hybrid molecule nano-array.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe molecule nano-array sensor, which is schematically shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, comprises a spot of hybrid molecules with each spot diameter of ~\u0026thinsp;100 nm (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Among them, the hybrid molecules consist of fluorescence-quencher molecules (F1\u003csup\u003e2+\u003c/sup\u003e), dye molecule RB, and cyclodextrin. F1\u003csup\u003e2+\u003c/sup\u003e molecule has wide fluorescence absorption capacity in the visible light range, especially in the 500\u0026ndash;650 and 700\u0026ndash;900 nm, which covers the excitation and emission wavelength of most dye molecules. RB dye molecule is selected as the fluorescence agent, a common dye molecule with absorption and emission center wavelengths of 530 nm and 590 nm, respectively, that can be quenched by F1\u003csup\u003e2+\u003c/sup\u003e molecule effectively. Cyclodextrin was used as a space barrier to prevent aggregation-caused quenching (ACQ) of dye molecules. The absorption spectra of the separated three molecules and their combination were shown in Figure S2a, which were characterized by spin-coating the corresponding molecules onto a cover glass. F1\u003csup\u003e2+\u003c/sup\u003e can be quickly reduced to an F2 molecule by H\u003csub\u003e2\u003c/sub\u003eS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), which has almost no absorption in the visible light range (Figure S2b). Based on this reaction platform, we printed the hybrid molecules with certain proportions into nano-arrays on the substrate by the modified nanoxerography method we developed.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e At each spot, the RB fluorescence emitted is quenched by F1\u003csup\u003e2+\u003c/sup\u003e molecules, so that the nano-arrays before reacting with H\u003csub\u003e2\u003c/sub\u003eS showed very weak fluorescence. When F1\u003csup\u003e2+\u003c/sup\u003e is decomposed by H\u003csub\u003e2\u003c/sub\u003eS molecules, the fluorescence of the RB regenerates, which can be easily imaged by a wide-field fluorescence microscope.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe fluorescent change of each spot in the nano-array correlates with the H\u003csub\u003e2\u003c/sub\u003eS concentration. We utilize an intensity integral (INTEN) and number counting (NUMB) method to quantify the fluorescence activities on the spots at high and low H\u003csub\u003e2\u003c/sub\u003eS concentrations, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e The INTEN method surveys all the spots in the nanoarray and integrates the fluorescence intensity, which can detect H\u003csub\u003e2\u003c/sub\u003eS concentration from 0.3 pM to 0.1 \u0026micro;M. At low concentrations, only parts of the spots have measurable fluorescence changes (defined as \u0026ldquo;on\u0026rdquo;). The NUMB method counts the separated \u0026ldquo;on\u0026rdquo; spots, showing a good correlation with the H\u003csub\u003e2\u003c/sub\u003eS concentration in the range of 1 fM to 100 pM, two orders of magnitude lower than that of INTEN method. The detailed configurations of INTEN and NUMB methods are listed in the Experimental section.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of hybrid molecule nano-array.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe molecule nano-array was fabricated by the modified nano-xerography method we had reported previously.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e The typical procedures are schematically shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea. First, a local surface charge array was written on the substrate by controlling a conductive AFM probe to apply high voltage (~ -80V), which can generate \u0026minus;\u0026thinsp;2.4 V charge potential on the substrate, as proved by the Kelvin probe force microscopy (KPFM) potential maps in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb. Then, F1\u003csup\u003e2+\u003c/sup\u003e, RB, and cyclodextrin molecules were dissolved in water as a dispersed phase to form water-in-oil reverse micelles, with hexane as a continuous phase and AOT as a surfactant. The micelles loaded with F1\u003csup\u003e2+\u003c/sup\u003e, RB, and cyclodextrin were spin-coated on the substrate. During this process, the molecules would be enriched and adsorbed onto the charged array. In the uncharged area, the perfluorinated substrate (CYTOP) could effectively avoid non-specific adsorption due to its ultra-low surface energy. As shown in AFM height map (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), a 5\u0026times;5 molecular nano-array with a spot size of ~\u0026thinsp;200 nm and a spacing of 5 \u0026micro;m was successfully assembled with less nonspecific adsorption. The micelle dispersion was characterized by dynamic light scattering (DLS) and Zeta potential measurement. The DLS result (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed) demonstrates the average micelle size in solution is around 3 nm, which is consistent with the results previously reported in the literature.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e The zeta potential is around \u0026minus;\u0026thinsp;31.1\u0026thinsp;\u0026plusmn;\u0026thinsp;11.0 mV, indicating the strong negative charge and the good stability of the micelles.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the steric spacer effect of cyclodextrin (CD) in the hybrid molecular system, we prepared RB micelles and RB/CD mixture micelles (1:1 molar ratio) and assembled them onto charged spots with identical surface potential. As expected, no fluorescence array was observed, attributed to aggregation-caused quenching (ACQ) of RB molecules (Figure S3a). Upon the addition of CD, a robust fluorescence nano-array emerged (Figure S3b). Subsequently, RB/CD was combined with the quencher molecule F1\u003csup\u003e2+\u003c/sup\u003e to form the final RB/CD/F1\u003csup\u003e2+\u003c/sup\u003e system (1:1:2 molar ratio). The average fluorescence intensity of the spots decreased significantly, reaching only 9% compared to the case without F1\u003csup\u003e2+\u003c/sup\u003e molecules (Figure S3c). Despite efforts to increase F1\u003csup\u003e2+\u003c/sup\u003e concentration, complete elimination of fluorescence proved challenging. This difficulty may arise from RB fluorescence being emitted in multiple directions, making it hard to complete quenching. Due to its promising characteristics, this RB/cyclodextrin/F1\u003csup\u003e2+\u003c/sup\u003e hybrid molecular system (1:1:2 molar ratio) was chosen for subsequent micro-area fluorescence detection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eTypical PL images of 5\u0026times;5 nano-array before and after reacting with H\u003csub\u003e2\u003c/sub\u003eS solution are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef. After the reaction, the fluorescence intensity of each hybrid molecule spot ranges from 300 to over 15000 arbitrary units (a.u.), compared to less than 290 a.u. of the negative controls (incubated with water). The fluorescence intensity on each spot is enhanced more than 50 times. These results clearly demonstrate the enormous potential of the nano-array in ultra-sensitive small molecule detection.\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantitatively H\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eS detection at high concentrations using INTEN method.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe quantificationally investigated the luminescence response of the hybrid molecule nano-array toward H\u003csub\u003e2\u003c/sub\u003eS. 10 \u0026micro;L of NaHS aqueous solution ranging from 0.1 fM to 1 \u0026micro;M were tested by the hybrid molecule nano-arrays (5\u0026times;5 square spots). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea shows the fluorescence images of the nano-array after sulfide assay from 10 pM to 1 \u0026micro;M. The images related to the other concentrations are shown in Figure S4. It is clear that the fluorescence intensity on each spot have a positive correlation with the concentration. All the spots are illuminated when the concentration is above 100 pM, on the contrary, only parts of spots are detectable in the images below 10 pM. To quantitatively analyze the correlation, we read out the fluorescence intensity of each spot. (Detailed information are shown in the Method section, and Figure S5-S6). If the intensity is larger than that before NaHS assay, we considered it an illuminated spot (\u0026ldquo;on\u0026rdquo; spot). The intensity sum of all the \u0026ldquo;on\u0026rdquo; spot versus corresponding concentration were plotted (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), noted as INTEN method. The plot is fitted with the well-established four-parameter logistic function curve (4-PL fitting), showing a good dynamic detection range over 5 magnitudes, from 300 fM to 1 \u0026micro;M. The linear range after logit transformation was between 100 pM and 100 nM, in which the spots are all reignites.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantitatively H\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eS detection at low concentrations using NUMB method.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs the NaHS concentration was below 10 pM, the sum of fluorescence intensity of the \u0026ldquo;on\u0026rdquo; spot is relatively low to quantitative analysis, and parts of the spots hold the \u0026ldquo;off\u0026rdquo; state (Figure S4). This is because not all the spots have the chance to react with hydrogen sulfide in several minutes at very low concentration. In view of this, we counted \u0026ldquo;on\u0026rdquo; spot numbers, and correlated them with the NaHS concentrations, noted as NUMB method. To get more reliable results at low concentration of NaHS, we increased the spot number to 400 by designing a 20\u0026times;20 array pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). NaHS solutions with concentrations from 0.1 fM to 1 nM were prepared. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea shows the fluorescence images corresponding to the concentration from 1fM to 100 pM, and all the \u0026ldquo;on\u0026rdquo; spots marked with circles. The 20\u0026times;20 molecule nano-array showed the NaHS detection limit of around 70 fM using INTEN method (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In comparison, the detection limit is about 300 fM with the same method when utilizing the 5\u0026times;5 nanoarray, indicating more spots can lead to a lower detection limit.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe images were further characterized by NUMB method to extend the NaHS detection limit to an even lower concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). After 4-PL fitting, a good correlation between the number of \u0026ldquo;on\u0026rdquo; spots and the concentration of NaHS from 1 fM to 100 pM is demonstrated with the \u003cem\u003eR\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e value of 0.99 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), indicating exceptional dynamic detection range. The detection limit has been reduced by about 2 orders of magnitude, compared to the INTEN method.\u003c/p\u003e \u003cp\u003eCombined with the results from \u003cb\u003eINTEN\u003c/b\u003e method at high NaHS concentrations and \u003cb\u003eNUMB\u003c/b\u003e method at low NaHS concentrations, this hybrid molecule nano-array platform has achieved the detection dynamic range from 1 fM to 0.1 \u0026micro;M, about 8 orders of magnitudes of response. This is a significant improvement in detection range and detection limit compared to the existing method for signaling small molecules.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe have developed a hybrid molecule nano-array sensor and implemented it for the highly sensitive detection of hydrogen sulfide. The nano-array features a small reaction volume, enabling a specific response from the F1\u003csup\u003e2+\u003c/sup\u003e molecule designed for H\u003csub\u003e2\u003c/sub\u003eS detection. With its high-sensitivity turn-on fluorescence detection mechanism, this nano-array achieves an exceptional detection limit of 1 fM using just a 10 \u0026micro;L solution. Furthermore, it exhibits a broad detection dynamic range spanning 8 orders of magnitudes. The outstanding performance of this nano-array is attributed to the robust nanoxerography technique and the use of well-developed molecular fluorescence probes. Notably, the versatility of this molecule nano-array extends beyond H\u003csub\u003e2\u003c/sub\u003eS detection, making it universally applicable to detect other signaling small molecules. This characteristic demonstrates its great potential in the bio-detection applications.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eChemicals and characterization.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSodium bis(2-ethylhexyl) sulfosuccinate (AOT, MACKLIIN, 96%), Rhodamine (RB, Aladdin, \u0026gt;\u0026thinsp;95.0% (HPLC)), β-Cyclodextrin (CD, Aladdin, 98%), Isooctane (Aladdin, 99%), cyclohexane (Shanghai Lingfeng Chemical Reagent Co. Ltd, \u0026ge;\u0026thinsp;99.7%, AR), DMSO (Sinopharm Chemical Reagent Co., Ltd, 99.0%), CYTOP (ASAHI GLASS COMPANY, Japan), F1\u003csup\u003e2+\u003c/sup\u003e (Deju Ye group, Nanjing University).\u003c/p\u003e \u003cp\u003eAll chemicals were used as received without further purification.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of reverse micelle solutions.\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e\u003c/h2\u003e \u003cp\u003eThe dye and F1\u003csup\u003e2+\u003c/sup\u003e stock solutions were prepared by dissolving dye molecule (RB or 1:1 RB/CD) in water and F1\u003csup\u003e2+\u003c/sup\u003e powders in water solution with a very small amount of DMSO, respectively. Then, RB/F1\u003csup\u003e2+\u003c/sup\u003e stock solution (molar ratio of 1:2) and RB/F1\u003csup\u003e2+\u003c/sup\u003e/CD stock solution (molar ratio of 1:2:1) were prepared with overall molecule concentration of 5.0 mM.\u003c/p\u003e \u003cp\u003eThe reverse micelle solutions were prepared by injecting 10 \u0026micro;L aqueous stock molecular solution into 82.6 \u0026micro;L AOT/isooctane solution with ultrasonication until forming clear and transparent solution. The concentration of AOT/isooctane was fixed at 1.12 M and thus the ω value of micelle solutions was kept at 6.0.\u003c/p\u003e \u003cp\u003eDynamic light scattering (DLS) and zeta potential measurements were performed on a 90 Plus/BI-MAS equipment (Brookhaven, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eFabrication of molecule nano-array.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e\u003c/h2\u003e \u003cp\u003eThe molecule nano-array was fabricated by modifying electric field-assisted surface sorption nano-printing (EFASP) method which consists of two stpes: charge array writing on a fluorine polymer electret (CYTOP) and micelle assembly on the charged array.\u003c/p\u003e \u003cp\u003eIn a typical process, fluorine polymer CYTOP was spin-coated on Si substrate which was pre-marked by lithography and baked to remove extra solvent for forming a 110 nm thick film (electret). Then, by applying high voltage (-40V to -90V) on a conductive AFM tip and writing charge on the electret film with pre-designed array pattern based on AFM scanning system, surface charge arrays (-0.15 to -3.0 V) were generated. At the same time, local surface modification occurs at the charged position, Kevin probe scanning (KPFM) measurements were determined whether the charge array was written successfully. All the charge writing process was performed in the air under ambient conditions (room temperature, relative humidity\u0026thinsp;=\u0026thinsp;20\u0026ndash;40%). The charge writing and KPFM scanning were performed on a NT-MDT AFM instrument.\u003c/p\u003e \u003cp\u003eAfter that, water-in-oil reverse micelle solution was dropped onto the charged area and tilted it after immersed 30s. Micelles containing hybrid molecules were captured to the charged arrays locally. The morphology after micelle assemblies is characterized by the AFM tapping mode scanning.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of hydrogen sulfide.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA thin layer of CTYOP (10\u0026ndash;30 nm) was spun on the assembled hybrid molecule nano-array to protect it from being rinsed off during the H\u003csub\u003e2\u003c/sub\u003eS detection. The CYTOP concentration, spin speed, spin time, and acceleration were set as 2%, 7000 rpm, 1 min and 2000 r/min\u003csup\u003e2\u003c/sup\u003e. There is no change in fluorescence image including fluorescence intensity and spots position before and after CYTOP covering.\u003c/p\u003e \u003cp\u003eNaHS aqueous solution with concentrations were first prepared ranging from 0.1 fM to 1 \u0026micro;M and used immediately after preparation (less than 5 minutes). During the detection, NaHS aqueous solutions (10 \u0026micro;L) with different concentrations were dropped onto different molecular nano-arrays and blown away by N\u003csub\u003e2\u003c/sub\u003e stream after 2 min. Finally, The nano-arrays were placed on a home-made wide-field inverted fluorescence microscope and their fluorescence photos were taken every 20 seconds.\u003c/p\u003e \u003cp\u003eThe fluorescence microscope was set up by Olympus inverted fluorescence microscope with PI spectrometer and EMCCD. The 532 continuous-wave laser is fiber-coupled and focused on the focal plane behind the objective lens through a long focal length lens. After passing through the 40\u0026times; objective lens, a spot of light about 120 microns in diameter focused on the sample. The emitted fluorescence is collected by an EMCCD after passing through the color filter with exposure time of 1s. The power of the excited light is fixed at 30 \u0026micro;W.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis.\u003c/h2\u003e \u003cp\u003eImage analysis was performed in MATLAB. Each original fluorescence image was calculated by subtracting the mean background value (M\u003csub\u003ebg\u003c/sub\u003e) and all the background (light intensity below 20 after subtracting M\u003csub\u003ebg\u003c/sub\u003e) was set to 0. We take these results as effective fluorescence images. Then we divided each effective fluorescence image into small images so that each small image contains one spot (Figure S5 and S7). We calculated the sum of fluorescence intensity (I\u003csub\u003esum\u003c/sub\u003e) of each spot. If the I\u003csub\u003esum\u003c/sub\u003e of a spot is larger than the average I\u003csub\u003esum\u003c/sub\u003e of spots before NaHS reaction, we considered it a illuminated spot (\u0026ldquo;on\u0026rdquo; spot). The average fluorescence intensity or the number of \u0026ldquo;on\u0026rdquo; spots were used to establish functional relationship with the concentration of NaHS, noted as INTEN and NUMB method, respectively.\u003c/p\u003e \u003cp\u003eCalibration curves were fit using a four-parameter logistic (4-PL) fit in Origin software. The values of fitting parameters are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. A linear regression was applied to the steepest part of the calibration curve. The background level is defined as the negative control (identical procedures using water instead of NaHS solutions) plusing three times of the standard deviation. The intersection of the regression line and the background level defined the LOD.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eNotes\u003c/h2\u003e \u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Key Technologies R\u0026amp;D Program of China (2022YFA1205602), grants from the National Natural Science Foundation of China (22075128), the Project funded by China Postdoctoral Science Foundation (No. BX2021124, 2021M701659). We acknowledge the assistance from the Technical Center of Nano Fabrication and Characterization, Nanjing University for the sample fabrication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSzabo, C. Hydrogen sulphide and its therapeutic potential. \u003cem\u003eNat. Rev. Drug. 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Synthesis of Au_Pd Bimetallic Nanoparticles in Reverse Micelles. \u003cem\u003eLangmuir\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 3877-3883 (2001).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"molecule nano-arrays, ultra-sensitive detection, hydrogen sulfide, fluorescence probe, nanoxerography","lastPublishedDoi":"10.21203/rs.3.rs-4455040/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4455040/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEarly disease diagnosis hinges on the sensitive detection of signaling molecules. Among these, hydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS) has emerged as a critical player in cardiovascular and nervous system signaling. On-chip immunoassays, particularly nanoarray-based interfacial detection, offer promising avenues for ultra-sensitive analysis due to their confined reaction volumes and precise signal localization. Beyond the DNA or protein biomolecules array, this work presents a promising hybrid small molecule nano-array for H\u003csub\u003e2\u003c/sub\u003eS detection, using the power of dual molecules: a dye for fluorescence emission and a quencher with specific H\u003csub\u003e2\u003c/sub\u003eS reactivity. Upon H\u003csub\u003e2\u003c/sub\u003eS interaction, the quenched fluorescence reignites, creating an easily detectable array of bright spots. The molecule nano-array sensor showed exceptional responses to H\u003csub\u003e2\u003c/sub\u003eS over 8 magnitudes of dynamic range from 1 fM to 0.1 μM, with a remarkable detection limit of 1 fM, just using a 10 μL solution. This new H\u003csub\u003e2\u003c/sub\u003eS detection method has the potential to significantly improve bioassay platforms, and the hybrid small-molecule nano-arrays we developed could be a valuable tool for advancing signaling molecule detection.\u003c/p\u003e","manuscriptTitle":"Ultra-Sensitive Hydrogen Sulfide Detection via Hybrid Small-Molecule Nano-arrays","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-07 05:40:51","doi":"10.21203/rs.3.rs-4455040/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e07e528a-983b-4827-9b98-c87812e329bc","owner":[],"postedDate":"June 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":32907324,"name":"Physical sciences/Nanoscience and technology/Nanobiotechnology/Nanofabrication and nanopatterning"},{"id":32907325,"name":"Physical sciences/Nanoscience and technology/Nanobiotechnology/Biosensors"},{"id":32907326,"name":"Physical sciences/Nanoscience and technology/Nanobiotechnology/Applications of AFM"}],"tags":[],"updatedAt":"2024-12-31T08:21:49+00:00","versionOfRecord":{"articleIdentity":"rs-4455040","link":"https://doi.org/10.1038/s41467-024-55123-y","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2024-12-30 05:00:00","publishedOnDateReadable":"December 30th, 2024"},"versionCreatedAt":"2024-06-07 05:40:51","video":"","vorDoi":"10.1038/s41467-024-55123-y","vorDoiUrl":"https://doi.org/10.1038/s41467-024-55123-y","workflowStages":[]},"version":"v1","identity":"rs-4455040","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4455040","identity":"rs-4455040","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}