Self-assembly of hyperbranched DNA network structure for signal amplification detection of miRNA.

Catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) can achieve the high sensitivity and rapid reaction rate in detecting miRNA. However, the amplification efficiency by these methods are limited. Herein, an enzyme-free and label-free hyperbranched DNA network structure (HDNS) was designed, in which localized catalytic hairpin assembly (LCHA) and hybridization chain reaction occurred in the horizontal axis and longitudinal axis, respectively, exhibiting intensive signal dual-amplification. miRNA-122 was selected as the target on behalf of miRNA to design the HDNS sensor. The fluorescence signal change of HDNS showed good linearity for detecting miRNA-122 in the concentration range from 0.1 nM to 60 nM with a limit of detection (LOD) at 37 pM which was lower than those of the sensors based on separate CHA or HCR. Afterwards, the HDNS sensor was applied to detect miRNA-122 in serum samples with the recovery rate in the range of 97.2 %-107 %. The sensor could distinguish different kinds of miRNAs, even the family members with high sequence homology, exhibiting excellent selectivity. This method provided a novel design strategy for improving the sensitivity and selectivity of DNA sensor for miRNA detection.

Isothermal Reciprocal Catalytic DNA Circuit for Sensitive Analysis of Kanamycin.

Inappropriate use of veterinary drugs can result in the presence of antibiotic residues in animal-derived foods, which is a threat to human health. A simple yet efficient antibiotic-sensing method is highly desirable. Programmable DNA amplification circuits have supplemented robust toolkits for food contaminants monitoring. However, they currently face limitations in terms of their intricate design and low signal gain. Herein, we have engineered a robust reciprocal catalytic DNA (RCD) circuit for highly efficient bioanalysis. The trigger initiates the cascade hybridization reaction (CHR) to yield plenty of repeated initiators for activating the rolling circle amplification (RCA) circuit. Then the RCA-generated numerous reconstituted triggers can reversely stimulate the CHR circuit. This results in a self-sufficient supply of numerous initiators and triggers for the successive cross-invasion of CHR and RCA amplifiers, thus leading to exponential signal amplification for the highly efficient detection of analytes. With its flexible programmability and modular features, the RCD amplifier can serve as a universal toolbox for the high-performance and accurate sensing of kanamycin in buffer and food samples including milk, honey, and fish, highlighting its enormous promise for low-abundance contaminant analysis in foodstuffs.

Triplex DNA-based aggregation-induced emission probe: A new platform for hybridization chain reaction-based fluorescence sensing assay.

The hybridization chain reaction (HCR), as one of the nucleic acid amplification technologies, is combined with fluorescence signal output with excellent sensitivity, simplicity, and stability. However, current HCR-based fluorescence sensing methods still have some defects such as the blocking effect of the HCR combination with fluorophores and the aggregation-caused quenching (ACQ) phenomenon of traditional fluorophores. Herein, a triplex DNA-based aggregation-induced emission probe (AIE-P) was designed as the fluorescent signal transduction, which is able to provide a new platform for HCR-based sensing assay. The AIE-P was synthesized by attaching the AIE fluorophores to terminus of the oligonucleotide through amido bond, and captured the products of HCR to form triplex DNA. In this case, the AIE fluorophores were located in close proximity to generate fluorescence. This assay provided turn-on fluorescence efficiency with a high signal-to-noise ratio and excellent amplification capability to solve the shortcoming of HCR-based fluorescence sensing methods. It enabled sensitive detection of Vibrio parahaemolyticus in the range of 102-106 CFU mL-1, and with a low limit of detection down to 39 CFU mL-1. In addition, this assay expressed good specificity and practicability. The triplex DNA-based AIE probe forms a universal molecular tool for developing HCR-based fluorescence sensing methods.

A label-free and immobilization-free approach for constructing photoelectrochemical nucleic acid sensors utilizing DNA-silver nanoparticle affinity interactions.

Efficient and affordable nucleic acid detection methods play a pivotal role in various applications. Herein, we developed an immobilization-free and label-free strategy to construct a photoelectrochemical nucleic acid biosensing platform based on interactions between silver nanoparticles and DNA. First, CRISPR-Cas12a exhibited a trans-cleavage effect on adenine nucleotide sequences upon recognizing the target DNA. The resulting adenine nucleotide sequences of varying lengths then engaged in interactions with silver nanoparticles, leading to a solution characterized by distinct light transmittance. Subsequently, the solution was positioned between the light source and the photoelectrode, strategically impacting the photon absorption step within the photoelectrochemical process. Consequently, the detection of nucleic acid was accomplished through the analysis of the resultant photocurrent signal. The developed platform exhibits a detection limit of 0.06 nM (S/N = 3) with commendable selectivity. The innovative use of adenine nucleotide sequences as cost-effective probes interacting with silver nanoparticles eliminates the need for complex interfacial immobilization processes, significantly simplifying the fabrication of DNA sensors. The outcomes of our research present a promising pathway for advancing the development of economically feasible miniature DNA sensors.

Signal amplification strategy of DNA self-assembled biosensor and typical applications in pathogenic microorganism detection.

Biosensors have emerged as ideal analytical devices for various bio-applications owing to their low cost, convenience, and portability, which offer great potential for improving global healthcare. DNA self-assembly techniques have been enriched with the development of innovative amplification strategies, such as dispersion-to-localization of catalytic hairpin assembly, and dumbbell hybridization chain reaction, which hold great significance for building biosensors capable of realizing sensitive, rapid and multiplexed detection of pathogenic microorganisms. Here, focusing primarily on the signal amplification strategies based on DNA self-assembly, we concisely summarized the strengths and weaknesses of diverse isothermal nucleic acid amplification techniques. Subsequently, both single-layer and cascade amplification strategies based on traditional catalytic hairpin assembly and hybridization chain reaction were critically explored. Furthermore, a comprehensive overview of the recent advances in DNA self-assembled biosensors for the detection of pathogenic microorganisms is presented to summarize methods for biorecognition and signal amplification. Finally, a brief discussion is provided about the current challenges and future directions of DNA self-assembled biosensors.

Simultaneous detection of dual microRNAs related to EV71 using ICP-MS based on metal nanoparticle labeling with hybridization chain reaction.

BACKGROUND: Hand, foot, and mouth (HMFD) disease caused by enterovirus 71 (EV 71), is closely associated with severe clinical manifestations and can be deadly. Early detection of EV 71 can be achieved by detecting the increment in miR296 and miR16 in the serum. Using HCR to amplify signals and convert biological signals into metal nanoparticle signals detectable by ICP-MS is a detection method that can collect more accurate and reliable information, compared with traditional methods, in the detection of biological samples. RESULTS: We described a strategy for the simultaneous detection of miR296 and miR16 by ICP-MS based on metal nanoparticles (NPs) labeling with HCR. Briefly, single-stranded DNA (ssDNA) and magnetic beads (MBs), as well as NPs and signal probes for miRNA (Sp-miR) were firstly conjugated via the streptavidin-biotin recognition system, constituting ssDNA-MBs and NPs-Sp-miR complex, respectively. The latter complex then hybridized with the former through HCR, generating the nanosensors for targets. Then, the targets were added and hybridized with ssDNA, and the HCR complex with NPs was released into the solution. Finally, the corresponding signals of the NPs were measured by ICP-MS. Results demonstrated that the developed method had good sensitivity and satisfactory selectivity and precision. Furthermore, when applied to biological samples with a complex matrix, the developed method also showed good recovery (88 % - 92 %) and reproducibility (RSD<10 %). SIGNIFICANCE: This method contributes to the early diagnosis of HFMD and opens up ideas for the further development of high-throughput biomarker detection. The strategy has practical potential for miR296 and miR16 detection in biological samples and provides a promising tool for multiple miRNA detection.

Bringing to Light the Importance of the miRNA Methylome in Colorectal Cancer Prognosis Through Electrochemical Bioplatforms.

This work reports the first electrochemical bioplatforms developed for the determination of the total contents of either target miRNA or methylated target miRNA. The bioplatforms are based on the hybridization of the target miRNA with a synthetic biotinylated DNA probe, the capture of the formed DNA/miRNA heterohybrids on the surface of magnetic microcarriers, and their recognition with an antibody selective to these heterohybrids or to the N6-methyladenosine (m6A) epimark. The determination of the total or methylated target miRNA was accomplished by labeling such secondary antibodies with the horseradish peroxidase (HRP) enzyme. In both cases, amperometric transduction was performed on the surface of disposable electrodes after capturing the resulting HRP-tagged magnetic bioconjugates. Because of their increasing relevance in colorectal cancer (CRC) diagnosis and prognosis, miRNA let-7a and m6A methylation were selected. The proposed electrochemical bioplatforms showed attractive analytical and operational characteristics for the determination of the total and m6A-methylated target miRNA in less than 75 min. These bioplatforms, innovative in design and application, were applied to the analysis of total RNA samples extracted from cultured cancer cells with different metastatic profiles and from paired healthy and tumor tissues of patients diagnosed with CRC at different stages. The obtained results demonstrated, for the first time using electrochemical platforms, the potential of interrogating the target miRNA methylation level to discriminate the metastatic capacities of cancer cells and to identify tumor tissues and, in a pioneering way, the potential of the m6A methylation in miRNA let-7a to serve as a prognostic biomarker for CRC.

Simultaneous detection of seven bacterial pathogens transmitted by flies using the reverse line blot hybridization assay.

BACKGROUND: Traditional methods for detecting insect-borne bacterial pathogens are time-consuming and require specialized laboratory facilities, limiting their applicability in areas without access to such resources. Consequently, rapid and efficient detection methods for insect-borne bacterial diseases have become a pressing need in disease prevention and control. METHODS: We aligned the ribosomal 16S rRNA sequences of seven bacterial species (Staphylococcus aureus, Shigella flexneri, Aeromonas caviae, Vibrio vulnificus, Salmonella enterica, Proteus vulgaris, and Yersinia enterocolitica) by DNASTAR Lasergene software. Using DNASTAR Lasergene and Primer Premier software, we designed universal primers RLB-F and RLB-R, two species-specific probes for each pathogen, and a universal probe (catch-all). The PCR products of seven standard strains were hybridized with specific oligonucleotide probes fixed on the membrane for specific experimental procedures. To evaluate the sensitivity of PCR-RLB, genomic DNA was serially diluted from an initial copy number of 1010 to 100 copies/µl in distilled water. These dilutions were utilized as templates for the PCR-RLB sensitivity analysis. Simultaneous detection of seven fly-borne bacterial pathogens from field samples by the established PCR-RLB method was conducted on a total of 1060 houseflies, collected from various environments in Lanzhou, China. RESULTS: The established PCR-RLB assay is capable of detecting bacterial strains of about 103 copies/µl for S. aureus, 103 copies/µl for S. flexneri, 105 copies/µl for A. caviae, 105 copies/µl for V. vulnificus, 100 copies/µl for S. enterica, 105 copies/µl for P. vulgaris, and 100 copies/µl for Y. enterocolitica. The results demonstrate that the detection rate of the established PCR-RLB method is higher (approximately 100 times) compared to conventional PCR. This method was applied to assess the bacterial carrier status of flies in various environments in Lanzhou, China. Among the seven bacterial pathogens carried by flies, S. enterica (34.57%), S. flexneri (32.1%), and Y. enterocolitica (20.37%) were found to be the predominant species. CONCLUSIONS: Overall, this research shows that the rapid and efficient PCR-RLB detection technology could be a useful for surveillance and therefore effective prevention and control the spread of insect-borne diseases. Meanwhile, the experimental results indicate that urban sanitation and vector transmission sources are important influencing factors for pathogen transmission.

Multiplex, Quantitative, High-Resolution Imaging of Protein:Protein Complexes via Hybridization Chain Reaction.

Signal amplification based on the mechanism of hybridization chain reaction (HCR) facilitates spatial exploration of gene regulatory networks by enabling multiplex, quantitative, high-resolution imaging of RNA and protein targets. Here, we extend these capabilities to the imaging of protein:protein complexes, using proximity-dependent cooperative probes to conditionally generate a single amplified signal if and only if two target proteins are colocalized within the sample. HCR probes and amplifiers combine to provide automatic background suppression throughout the protocol, ensuring that even if reagents bind nonspecifically in the sample, they will not generate amplified background. We demonstrate protein:protein imaging with a high signal-to-background ratio in human cells, mouse proT cells, and highly autofluorescent formalin-fixed paraffin-embedded (FFPE) human breast tissue sections. Further, we demonstrate multiplex imaging of three different protein:protein complexes simultaneously and validate that HCR enables accurate and precise relative quantitation of protein:protein complexes with subcellular resolution in an anatomical context. Moreover, we establish a unified framework for simultaneous multiplex, quantitative, high-resolution imaging of RNA, protein, and protein:protein targets, with one-step, isothermal, enzyme-free HCR signal amplification performed for all target classes simultaneously.

Heteromultivalency enables enhanced detection of nucleic acid mutations.

Detecting genetic mutations such as single nucleotide polymorphisms (SNPs) is necessary to prescribe effective cancer therapies, perform genetic analyses and distinguish similar viral strains. Traditionally, SNP sensing uses short oligonucleotide probes that differentially bind the SNP and wild-type targets. However, DNA hybridization-based techniques require precise tuning of the probe's binding affinity to manage the inherent trade-off between specificity and sensitivity. As conventional hybridization offers limited control over binding affinity, here we generate heteromultivalent DNA-functionalized particles and demonstrate optimized hybridization specificity for targets containing one or two mutations. By investigating the role of oligo lengths, spacer lengths and binding orientation, we reveal that heteromultivalent hybridization enables fine-tuned specificity for a single SNP and dramatic enhancements in specificity for two non-proximal SNPs empowered by highly cooperative binding. Capitalizing on these abilities, we demonstrate straightforward discrimination between heterozygous cis and trans mutations and between different strains of the SARS-CoV-2 virus. Our findings indicate that heteromultivalent hybridization offers substantial improvements over conventional monovalent hybridization-based methods.

Target-triggered dual signal amplification based on HCR-enhanced nanozyme activity for the sensitive visual detection of Escherichia coli.

The detection of foodborne pathogens is crucial for food hygiene regulation and disease diagnosis. Colorimetry has become one of the main analytical methods in studying foodborne pathogens due to its advantages of visualization, low cost, simple operation, and no complex instrument. However, the low sensitivity limits its applications in early identification and on-site detection for trace analytes. In order to overcome such a limitation, herein we propose a joint strategy featuring dual signal amplification based on the hybridization chain reaction (HCR) and DNA-enhanced peroxidase-like activity of gold nanoparticles (AuNPs) for the sensitive visual detection of Escherichia coli. Target bacteria bound specifically to the aptamer domain in the capture hairpin probe, exposing the trigger domain for HCR and forming the extended double-stranded DNA (dsDNA) structures. The peroxidase-like catalytic capacity of AuNPs can be enhanced significantly by dsDNAs with the sticky ends of dsDNAs being adsorbed on AuNPs and the rigidity of dsDNAs causing the spatial regulation of AuNP concentration. The intensity of the enhancement was linearly related to the number of target bacteria. With the above strategy, the detection limit of our colorimetric method for Escherichia coli was down to 28 CFU mL-1 within a short analytical time (50 min). This study provides a new perspective for the sensitive and visual detection of early bacterial contamination in foods.

Selective miR-21 detection technology based on photocrosslinkable artificial nucleic acid-modified magnetic particles and hybridization chain reaction.

Recently, microRNA (miRNA) detection in blood has attracted attention as a new early detection technology for cancer. The extraction of target miRNA is a necessary preliminary step for detection; however, currently, most extraction methods extract all RNA from the blood, which limits the detection selectivity. Therefore, a method for the selective extraction and detection of target miRNA from blood is very important. In this study, we utilized photocrosslinkable artificial nucleic acids and the hybridization chain reaction (HCR) in an attempt to improve upon the current standard method RT-qPCR, which is hampered by problems with primer design and enzymatic amplification. By introducing photocrosslinkable artificial nucleic acids to oligonucleotide probes modified with magnetic particles with a sequence complementary to that of the target miRNA and irradiating them with light, covalent bonds were formed between the target miRNA and the oligonucleotide probes. These tight covalent bonds enabled the capture of miRNA in blood, and intensive washing ensured that only the target miRNA were extracted. After extraction, two types of DNA (H1 and H2) modified with fluorescent dyes were added and the fluorescence signals were amplified by the HCR in the presence of the target miRNA bound to the photocrosslinkable artificial nucleic acids, allowing for isothermal and enzyme-free miRNA detection. The novel method is suitable for selective miRNA detection in real blood samples. Because the reaction proceeds isothermally and no specialized equipment is used for washing, this detection technology is simple and selective and suitable for application to point-of-care technology using microfluidic devices.

Genetically engineered virus-like particle-armoured and multibranched DNA scaffold-corbelled ultra-sensitive hierarchical hybridization chain reaction for targeting-enhanced imaging in living biosystems under spatiotemporal light powering.

Although nucleic acids-based fluorescent biosensors, exemplified by the hybridization chain reaction (HCR), have exhibited promise as an imaging tool for detecting disease-related biomolecular makers in living biosystems, they still face certain challenges. These include the need for improved sensitivity, poor bio-targeting capability, the absence of signal enrichment interface and the uncontrollable biosensing initiation. Herein, we present a range of effective solutions. First, a stacking design resembling building blocks is used to construct a special hierarchical HCR (termed H-HCR), for which a hierarchical bridge is employed to graft multiunit HCR products. Furthermore, the H-HCR components are encapsulated into a virus-like particle (VLP) endowed with a naturally peptide-mediated targeting unit through genetic engineering of plasmids, after which the biosensor can specifically identify cancer cytomembranes. By further creating a multibranched DNA scaffold to enrich the H-HCR produced detection signals, the biosensor's analyte recognition module is inserted with a photocleavage-linker, allowing that the biosensing process can be spatiotemporally initiated via a light-powered behavior. Following these innovations, this genetically engineered VLP-armoured and multibranched DNA-scaffold-corbelled H-HCR demonstrates an ultra-sensitive and specific biosensing performance to a cancer-associated microRNA marker (miRNA-155). Beyond the worthy in vitro analysis, our method is also effective in performing imaging assays for such low-abundance analyte in living cells and even bodies, thus providing a roust platform for disease diagnosis.

Imaging mRNA in vitro and in vivo with nanofirecracker probes via intramolecular hybridization chain reaction.

Hybridization chain reaction (HCR) based enzyme-free amplification techniques have recently been developed for the visualization of intracellular messenger RNA (mRNA). However, the slow kinetics and potential interference with the intricate biological environments hinder its application in the clinic and in vivo. Herein, we designed a nanofirecracker probe-based strategy using intramolecular hybridization chain reaction (IHCR) amplifier for rapid, efficient, sensitive, specific detection and imaging of survivin mRNA both in vitro and vivo. Two probes, HP1 and HP2, in IHCR were simultaneously incorporated into a DNA nanowire scaffolds to bring HP1 and HP2 to close proximity on the assembled nanowire scaffolds. Empowered by the DNA nanowire scaffolds and spatial confinement effect, the nanofirecracker probe-based IHCR sensing system exhibited improved biostability, accelerated reaction kinetics, and enhanced signal amplification. This new strategy has been successfully applied to imaging mRNA in both cultured cells and in mice. Importantly, this novel sensing method was capable of detecting survivin mRNA in clinical blood samples from subjects with colorectal cancer. Thus, this novel nanofirecracker probe-based IHCR strategy holds great potential in advancing both biomedical research and in molecular diagnostics.

Recent advances in optical biosensing and imaging of telomerase activity and relevant signal amplification strategies.

Telomerase as a new valuable biomarker for early diagnosis and prognosis evaluation of cancer has attracted much interest in the field of biosensors, cell imaging, and drug screening. In this review, we mainly focus on different optical techniques and various signal amplification strategies for telomerase activity determination. Fluorometric, colorimetry, chemiluminescence, surface-enhanced Raman scattering (SERS), and dual-mode techniques for telomerase sensing and imaging are summarized. Signal amplification strategies include two categories: one is nucleic acid-based amplification, such as rolling circle amplification (RCA), the hybridization chain reaction (HCR), and catalytic hairpin assembly (CHA); the other is nanomaterial-assisted amplification, including metal nanoclusters, quantum dots, transition metal compounds, graphene oxide, and DNA nanomaterials. Challenges and prospects are also discussed to provide new insights for future development of multifunctional strategies and techniques for in situ and in vivo analysis of biomarkers for accurate cancer diagnosis.

Plasmonic Probing of Deoxyribonucleic Acid Hybridization at the Single Base Pair Resolution.

Partial DNA duplex formation greatly impacts the quality of DNA hybridization and has been extensively studied due to its significance in many biological processes. However, traditional DNA sensing methods suffer from time-consuming amplification steps and hinder the acquisition of information about single-molecule behavior. In this work, we developed a plasmonic method to probe the hybridization process at a single base pair resolution and study the relationship between the complementarity of DNA analytes and DNA hybridization behaviors. We measured single-molecule hybridization events with Au NP-modified ssDNA probes in real time and found two hybridization adsorption events: stable and transient adsorption. The ratio of these two hybridization adsorption events was correlated with the length of the complementary sequences, distinguishing DNA analytes from different complementary sequences. By using dual incident angle excitation, we recognized different single-base complementary sequences. These results demonstrated that the plasmonic method can be applied to study partial DNA hybridization behavior and has the potential to be incorporated into the identification of similar DNA sequences, providing a sensitive and quantitative tool for DNA analysis.

Engineering of a Self-Regulatory Bidirectional DNA Assembly Circuit for Amplified MicroRNA Imaging.

The engineering of catalytic hybridization DNA circuits represents versatile ways to orchestrate a complex flux of molecular information at the nanoscale, with potential applications in DNA-encoded biosensing, drug discovery, and therapeutics. However, the diffusive escape of intermediates and unintentional binding interactions remain an unsolved challenge. Herein, we developed a compact, yet efficient, self-regulatory assembly circuit (SAC) for achieving robust microRNA (miRNA) imaging in live cells through DNA-templated guaranteed catalytic hybridization. By integrating the toehold strand with a preblocked palindromic fragment in the stem domain, the proposed miniature SAC system allows the reactant-to-template-controlled proximal hybridization, thus facilitating the bidirectional-sustained assembly and the localization-intensified signal amplification without undesired crosstalk. With condensed components and low reactant complexity, the SAC amplifier realized high-contrast intracellular miRNA imaging. We anticipate that this simple and template-controlled design can enrich the clinical diagnosis and prognosis toolbox.

Construction and bioanalytical applications of poly-adenine-mediated gold nanoparticle-based spherical nucleic acids.

Owing to the versatile photophysical and chemical properties, spherical nucleic acids (SNAs) have been widely used in biosensing. However, traditional SNAs are formed by self-assembly of thiolated DNA on the surface of a gold nanoparticle (AuNP), where it is challenging to precisely control the orientation and surface density of DNA. As a new SNA, a polyadenine (polyA)-mediated SNA using the high binding affinity of consecutive adenines to AuNPs shows controllable surface density and configuration of DNA, which can be used to improve the performance of a biosensor. Herein, we first introduce the properties of polyA-mediated SNAs and fundamental principles regarding the polyA-AuNP interaction. Then, we provide an overview of current representative synthesis methods of polyA-mediated SNAs and their advantages and disadvantages. After that, we summarize the application of polyA-mediated SNAs in biosensing based on fluorescence and colorimetric methods, followed by discussion and an outlook of future challenges in this field.

A Canvas of Spatially Arranged DNA Strands that Can Produce 24-bit Color Depth.

Nucleic acid microarray photolithography combines density, throughput, and positional control in DNA synthesis. These surface-bound sequence libraries are conventionally used in large-scale hybridization assays against fluorescently labeled, perfect-match DNA strands. Here, we introduce another layer of control for in situ microarray synthesis─hybridization affinity─to precisely modulate fluorescence intensity upon duplex formation. Using a combination of Cy3-, Cy5-, and fluorescein-labeled targets and an ensemble of truncated DNA probes, we organize 256 shades of red, green, and blue intensities that can be superimposed and merged. In so doing, hybridization alone is able to produce a large palette of 16 million colors or 24-bit color depth. Digital images can be reproduced with high fidelity at the micrometer scale by using a simple process that assigns sequence to any RGB value. Largely automated, this approach can be seen as miniaturized DNA-based painting.

Dipodal Silanes Greatly Stabilize Glass Surface Functionalization for DNA Microarray Synthesis and High-Throughput Biological Assays.

Glass is by far the most common substrate for biomolecular arrays, including high-throughput sequencing flow cells and microarrays. The native glass hydroxyl surface is modified by using silane chemistry to provide appropriate functional groups and reactivities for either in situ synthesis or surface immobilization of biologically or chemically synthesized biomolecules. These arrays, typically of oligonucleotides or peptides, are then subjected to long incubation times in warm aqueous buffers prior to fluorescence readout. Under these conditions, the siloxy bonds to the glass are susceptible to hydrolysis, resulting in significant loss of biomolecules and concomitant loss of signal from the assay. Here, we demonstrate that functionalization of glass surfaces with dipodal silanes results in greatly improved stability compared to equivalent functionalization with standard monopodal silanes. Using photolithographic in situ synthesis of DNA, we show that dipodal silanes are compatible with phosphoramidite chemistry and that hybridization performed on the resulting arrays provides greatly improved signal and signal-to-noise ratios compared with surfaces functionalized with monopodal silanes.