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The conventional hybridization chain reaction (HCR)-based electrochemical biosensors usually require the immobilization of probes on the electrode surface. This will limit the applications of biosensors due to the shortcomings of complex immobilization processes and low HCR efficiency. In this work, we proposed astrategy for the design of HCR-based electrochemical biosensors by integrating the advantages of homogeneous reaction and heterogeneous detection. Specifically, the targets triggered the autonomous cross-opening and hybridization oftwobiotin-labeled hairpin probes to form long-nicked dsDNA polymers. The HCR products with many biotin tags were then captured by a streptavidin-covered electrode, thus allowing for the attachment of streptavidin-conjugated signal reporters through streptavidin–biotin interactions. By employing DNA and microRNA-21 as the model targets and glucose oxidase as the signal reporter, the analytical performances of the HCR-based electrochemical biosensors were investigated. The detection limits of this method were found to be 0.6 fM and 1 fM for DNA and microRNA-21, respectively. The proposed strategy exhibited good reliability for target analysis in serum and cellular lysates. The strategy can be used to develop various HCR-based biosensors for a wide range of applications because sequence-specific oligonucleotides exhibit high binding affinity to a series of targets. In light of the high stability and commercial availability of streptavidin-modified materials, the strategy can be used for the design of different biosensors by changing the signal reporter and/or the sequence of hairpin probes.

[EN] Massive DNA testing requires novel technologies to support a sustainable health system. In recent years, DNA superstructures have emerged as alternative probes and transducers. We, herein, report a multiplexed and highly sensitive approach based on an allele-specific hybridization chain reaction (AS-HCR) in the array format to detect single-nucleotide variants. Fast isothermal amplification was developed before activating the HCR process on a chip to work with genomic DNA. The assay principle was demonstrated, and the variables for integrating the AS-HCR process and smartphone-based detection were also studied. The results were compared to a conventional polymerase reaction chain (PCR)-based test. The developed multiplex method enabled higher selectivity against single-base mismatch sequences at concentrations as low as 103 copies with a limit of detection of 0.7% of the mutant DNA percentage and good reproducibility (relative error: 5% for intra-assay and 17% for interassay). As proof of concept, the AS-HCR method was applied to clinical samples, including human cell cultures and biopsied tissues of cancer patients. Accurate identification of single-nucleotide mutations in KRAS and NRAS genes was validated, considering those obtained from the reference sequencing method. To conclude, AS-HCR is a rapid, simple, accurate, and cost-effective isothermal method that detects clinically relevant genetic variants and has a high potential for point-of-care demands., The authors acknowledge the financial support received from EU FEDER, the Spanish Ministry of Economy and Competitiveness (PID2019-110713RB-I00), and the Generalitat Valenciana (PROMETEO/2020/094 and GVA-FPI-2017 Ph.D. grant).

In the continuous combat against diseases, there is the need for tools that enable an improved diagnostic efficiency towards higher information density combined with reduced time-to-result and cost. Here, a novel fully integrated microfluidic platform, the Evalution™, is evaluated as a potential solution to this need. Encoded microparticles combined with channel-based microfluidics allow a fast, sensitive and simultaneous detection of several disease-related biomarkers. Since the binary code is represented by physically present holes, 210 different codes can be created that will not be altered by light or chemically induced degradation. Exploiting the unique features of this multiplex platform, hybridization chain reaction (HCR) is explored as a generic approach to reach the desired sensitivity. Compared to a non-amplified reference system, the sensitivity was drastically improved by a factor of 104, down to low fM LOD values. Depending on the HCR duration, the assay can be tuned for sensitivity or total assay time, as desired. The huge potential of this strategy was further demonstrated by the successful detection of a multiplex panel of six different nucleic acid targets including viruses and bacteria. The ability to not only discriminate these two categories but, with the same effort, also virus strains (human adenovirus and human bocavirus), virus subtypes (human adenovirus type B and D) and antibiotic-resistant bacteria (Streptococcus pneumonia), exemplifies the specificity of the developed approach. The effective, yet highly simplified, isothermal and protein-enzyme-free signal amplification tool reaches an LOD ranging from as low as 33 ± 4 to 151 ± 12 fM for the different targets. Moreover, direct detection in a clinically relevant sample matrix was verified, resulting in a detection limit of 309 ± 80 fM, approximating the low fM levels detectable with the gold standard analysis method, PCR, without the drawbacks related to protein enzymes, thermal cycling and elaborate sample preparation steps. The reported strategy can be directly transferred as a generic approach for the sensitive and specific detection of various target molecules in multiplex. In combination with the high-throughput capacity and reduced reagent consumption, the Evalution™ demonstrates immense potential in the next generation of diagnostic tools towards more personalized medicine. ispartof: Biosensors vol:13 issue:1 ispartof: location:Switzerland status: Published online

Cellular functions are governed by intricate chains of interactions between proteins. In order to properly understand cellular biology one must look into, not only protein function, but also its interacting network. Furthermore, due to the large heterogeneity between cells within cultures and tissue samples it is important to retain spatial information to enable investigations on a single cell level. In order to achieve this, in situ methods play a large importance in further elucidation of these interacting networks. In order to investigate interactions between macromolecules such as proteins and nucleic acids, many outstanding methods have been developed. Some focusing on larger scale analysis, some on live cell imaging and some on detecting novel interactions. Our own group has focused on in situ methods utilizing DNA conjugated antibodies. DNA itself is a great macromolecule to work with, it can be produced synthetically, DNA hybridization is highly predictable and there is a large repertoire of DNA-modifying enzymes. This has been used in the development of methods such as proximity ligation assay (PLA) and Proximity-dependent initiation of hybridization chain reaction (ProxHCR). Both methods utilize antibodies conjugated with DNA in order to detect proximity events between two proteins. PLA utilizes ligation to confirm proximity, while ProxHCR utilizes a chain of strand displacements to do the same. Both methods work well, but no method is beyond further optimization. For PLA, a general concern lies in the formation of incorrectly interacting probes, resulting in incorrect ligations that yield linear fragments, incapable of producing visible signal. As a result PLA can produce a substantial amount of false negatives. To address this, we produced a similar method, Unfold, to streamline the probe interactions and ligations to improve efficiency. For ProxHCR the original method required overly stringent reactions conditions to allow for efficient strand displacements and thus strong signal. Furthermore, signal strength was further compromised by oligonucleotide quality. To improve these issues, the ProxHCR method was completely redesigned and oligonucleotide quality along with signal strength was improved by further purification. Both optimizations resulted in more efficient and versatile methods suitable for routine lab work and potential diagnostic use.

Hybridization chain-reaction (HCR) is technique to generate a linear polymerization of oligonucleotide hairpins, used in multiple molecular biology methods. The HCR reaction is dependent on that every hairpin is metastable in the absence of a triggering oligonucleotide and that every hairpin can continue the polymerization, which places a strong demand on oligonucleotide quality. In this paper we show how further purification can greatly increase polymerization potential. We found that a single extra PAGE-purification could greatly enhance hairpin polymerization both in solution and in situ. Purification using a ligation-based method further improved polymerization, yielding in situ immunoHCR stains at least 3.4-times stronger than non-purified control. This demonstrates the importance of not only good sequence design of the oligonucleotide hairpins, but also the demand for high quality oligonucleotides to accomplish a potent and specific HCR.

In this study, Lba Cas12a (Cpf1) as one of the CRISPR systems from Lachnospiraceae bacterium was coupled with a hybridization chain reaction (HCR) to develop an electrochemical biosensor for detecting the pathogenic bacterium, Salmonella typhimurium. Autonomous cross-opening of functional DNA hairpin structures of HCR yielded polymer double-stranded DNA wires consisting of numerous single-stranded DNAs, which initiated the trans-cleavage activity of CRISPR-Cas12a to indiscriminately cleave random single-stranded DNA labeling electrochemical tags on the surface of the electrode. It led to a variation in the electron transfer of electrochemical tags. The polymer double-stranded DNA of HCR was immobilized on dynabeads (DBs) via the S. typhimurium aptamer and released from DBs. The established method could selectively and sensitively quantify S. typhimurium in samples with detection limits of 20 CFU/mL. Our study provides a novel insight for exploring universal analytical methods for pathogenic bacteria based on CRISPR-Cas12a coupled with HCR. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03733-6.

Cancer immunotherapy has made recent breakthrough, including immune checkpoint blockade (ICB) that inhibits immunosuppressive checkpoints such as programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1). However, most cancer patients do not durably respond to ICB. To predict ICB responses for patient stratification, conventional immunostaining has been used to analyze the PD-L1 expression level on biopsied tumor tissues but has limitations of invasiveness and tumor heterogeneity. Recently, PD-L1 levels on tumor cell exosomes showed the potential to predict ICB response. Here, we developed a non-invasive, sensitive, and fast assay, termed as exosome-hybridization chain reaction (ExoHCR), to analyze tumor cell exosomal PD-L1 levels. First, using αCD63-conjugated magnetic beads, we isolated exosomes from B16F10 melanoma and CT26 colorectal cancer cells that were immunostimulated to generate PD-L1-positive exosomes. Exosomes were then incubated with a conjugate of PD-L1 antibody with an HCR trigger DNA (T), in which one αPD-L1-T conjugate carried multiple copies of T. Next, a pair of metastable fluorophore-labeled hairpin DNA (H1 and H2) were added, allowing T on αPD-L1-T to initiate HCR in situ on bead-conjugated exosome surfaces. By flow cytometric analysis of the resulting beads, relative to αPD-L1-fluorophore conjugates, ExoHCR amplified the fluorescence signal intensities for exosome detection by 3–7 times in B16F10 cells and CT26 cells. Moreover, we validated the biostability of ExoHCR in culture medium supplemented with 50% FBS. These results suggest the potential of ExoHCR for non-invasive, sensitive, and fast PD-L1 exosomal profiling in patient stratification of cancer immunotherapy.

Assays for the high throughput screening of crops for virus monitoring need to be quick, easy, and low cost. One method involves using tissue blot immunoassays (TBIA), where plant stems are blotted onto nitrocellulose membrane and screened with available antibodies against a range of viruses. TBIAs are inexpensive but limited by antibody availability and specificity. To circumvent the antibody limitations, we developed the tissue blot hybridization chain reaction (TB-HCR). As with TBIA, plant stems are blotted onto a nitrocellulose membrane, however, TB-HCR involves using nucleic acid probes instead of antibodies. We demonstrated for the first time that TB-HCR can be used for plant viruses by designing and testing probes against species from several virus genera including begomovirus, polerovirus, luteovirus, cucumovirus, and alfamovirus. We also explored different hairpin reporter methods such as biotin/streptavidin-AP and the Alexa Fluor-488 Fluorophore. TB-HCR has applications for low-cost diagnostics for large sample numbers, rapid diagnostic deployment for new viruses, and can be performed as a preliminary triage assay prior to downstream applications.

Tumor-associated cell-free DNA (cfDNA) is a dynamic biomarker for genetic analysis, early diagnosis and clinical treatment of cancers. However, its detection has limitations because of its low abundance in blood or other complex bodily fluids. Herein, we developed an ultrasensitive cfDNA electrochemical biosensor (E-cfDNA sensor) based on tetrahedral DNA framework (TDF)-modified gold nanoparticles (Au NPs) with an interface for cfDNA detection. By accurately controlling the numbers of base pairs on each DNA framework, three types of TDFs were programmed: 26 base pairs of TDF; 17 base pairs of TDF; and 7 base pairs of TDF (TDF-26, TDF-16 and TDF-7, respectively). We also combined the TDF with hybridization chain reaction (HCR) to achieve signal amplification. Under optimal conditions, we detected the breast cancer susceptibility gene 1 (BRCA-1), a representative cfDNA closely related to breast cancer. An ultra-low detection limit of 1 aM with a linear range from 1 aM to 1 pM by TDF-26 was obtained, which was superior to the existing methods. Each type of TDF has excellent discrimination ability, which can distinguish single mismatch. More significantly, we also detected BRCA-1 in mimic serum samples, demonstrating that the E-cfDNA sensor has potential use in clinical research.

Zhiyuan Xu,1 Ronghua Ni,1 Yun Chen1,21School of Pharmacy, Nanjing Medical University, Nanjing 211166, People’s Republic of China; 2State Key Laboratory of Reproductive Medicine, Nanjing 210029, People’s Republic of ChinaCorrespondence: Yun ChenSchool of Pharmacy, Nanjing Medical University, 818 Tian Yuan East Road, Nanjing 211166, People’s Republic of ChinaTel +86 258 686 8326Fax +86 258 686 8467Email ychen@njmu.edu.cnBackground: Cancer relapse and metastasis is an obstacle to the treatment of breast cancer. Breast cancer stem cells (BCSCs), which can evade the killing effect of traditional chemotherapies, such as doxorubicin (DOX), may contribute to cancer development. Therefore, it is necessary to develop novel drugs that can target and eliminate BCSCs. While multiple strategies have been conceived, they are normally limited by the low drug loading capacity.Purpose: An aptamer-conjugated DNA nanotrain TA6NT-AKTin-DOX, which consists of a CD44 aptamer TA6, DNA building blocks M1 and M2 conjugated with an AKT inhibitor peptide AKTin individually and DOX, was designed.Methods: This DNA nanotrain was prepared through hybridization chain reactionand this highly ordered DNA duplex has plenty of sites where DOX and AKTin can be intercalated or anchored. By performing on MCF-7 BCSCs and tumors by xenografting BCSCs into nude mice, efficacy of the newly prepared drug was evaluated and compared with that of free DOX and various DNA nanotrains.Results: TA6NT-AKTin-DOX showed better efficacy both in vitro and in vivo. To some extent, the enhanced efficacy could be attributed to the targeting effect of TA6 and the high drug loading capacity of the nanotrain (∼20 DOX molecules). Besides, a synergistic response was demonstrated by combining DOX with AKTin, probably due to that the anchored AKTin can reverse the drug resistance of BCSCs including apoptosis resistance and ABC transporters overexpression via the AKT signaling pathway.Conclusion: The aptamer-conjugated DNA nanotrain TA6NT-AKTin-DOX demonstrated its targeting capability to BCSCs.Keywords: breast cancer stem cells (BCSCs), aptamer-conjugated DNA nanotrain, CD44 aptamer TA6, doxorubicin (DOX), AKT peptide inhibitor, hybridization chain reaction

Zhiyuan Xu,1 Ronghua Ni,1 Yun Chen1,21School of Pharmacy, Nanjing Medical University, Nanjing 211166, People’s Republic of China; 2State Key Laboratory of Reproductive Medicine, Nanjing 210029, People’s Republic of ChinaCorrespondence: Yun ChenSchool of Pharmacy, Nanjing Medical University, 818 Tian Yuan East Road, Nanjing 211166, People’s Republic of ChinaTel +86 258 686 8326Fax +86 258 686 8467Email ychen@njmu.edu.cnBackground: Cancer relapse and metastasis is an obstacle to the treatment of breast cancer. Breast cancer stem cells (BCSCs), which can evade the killing effect of traditional chemotherapies, such as doxorubicin (DOX), may contribute to cancer development. Therefore, it is necessary to develop novel drugs that can target and eliminate BCSCs. While multiple strategies have been conceived, they are normally limited by the low drug loading capacity.Purpose: An aptamer-conjugated DNA nanotrain TA6NT-AKTin-DOX, which consists of a CD44 aptamer TA6, DNA building blocks M1 and M2 conjugated with an AKT inhibitor peptide AKTin individually and DOX, was designed.Methods: This DNA nanotrain was prepared through hybridization chain reactionand this highly ordered DNA duplex has plenty of sites where DOX and AKTin can be intercalated or anchored. By performing on MCF-7 BCSCs and tumors by xenografting BCSCs into nude mice, efficacy of the newly prepared drug was evaluated and compared with that of free DOX and various DNA nanotrains.Results: TA6NT-AKTin-DOX showed better efficacy both in vitro and in vivo. To some extent, the enhanced efficacy could be attributed to the targeting effect of TA6 and the high drug loading capacity of the nanotrain (∼20 DOX molecules). Besides, a synergistic response was demonstrated by combining DOX with AKTin, probably due to that the anchored AKTin can reverse the drug resistance of BCSCs including apoptosis resistance and ABC transporters overexpression via the AKT signaling pathway.Conclusion: The aptamer-conjugated DNA nanotrain TA6NT-AKTin-DOX demonstrated its targeting capability to BCSCs.Keywords: breast cancer stem cells (BCSCs), aptamer-conjugated DNA nanotrain, CD44 aptamer TA6, doxorubicin (DOX), AKT peptide inhibitor, hybridization chain reaction

The study of gene expression in fungi has typically relied on measuring transcripts in populations of cells. A major disadvantage of this approach is that the transcripts’ spatial distribution and stochastic variation among individual cells within a clonal population is lost. Traditional fluorescence in situ hybridization techniques have been of limited use in fungi due to poor specificity and high background signal. Here, we report that in situ hybridization chain reaction (HCR), a method that employs split-initiator probes to trigger signal amplification upon mRNA-probe hybridization, is ideally suited for the imaging and quantification of low-abundance transcripts at single-cell resolution in the fungus Candida albicans. We show that HCR allows the absolute quantification of transcripts within a cell by microscopy as well as their relative quantification by flow cytometry. mRNA imaging also revealed the subcellular localization of specific transcripts. Furthermore, we establish that HCR is amenable to multiplexing by visualizing different transcripts in the same cell. Finally, we combine HCR with immunostaining to image specific mRNAs and proteins simultaneously within a single C. albicans cell. The fungus is a major pathogen in humans where it can colonize and invade mucosal surfaces and most internal organs. The technical development that we introduce, therefore, paves the way to study the patterns of expression of pathogenesis-associated C. albicans genes in infected organs at single-cell resolution. IMPORTANCE Tools to visualize and quantify transcripts at single-cell resolution have enabled the dissection of spatiotemporal patterns of gene expression in animal cells and tissues. Yet the accurate quantification of transcripts at single-cell resolution remains challenging for the much smaller microbial cells. Widespread phenomena such as stochastic variation in transcript levels among cells—even within a clonal population—seem to play important roles in the biology of many microorganisms. Investigating this process requires microbial cell-optimized procedures to image and measure mRNAs at single-molecule resolution. In this report, we adapt and expand in situ hybridization chain reaction (HCR) combined with split-initiator probes to visualize transcripts in the human-pathogenic fungus Candida albicans at high resolution.

The continuing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which causes coronavirus disease 2019 (COVID-19), has spread globally and its reliable diagnosis is one of the foremost priorities for protecting public health. Herein a rapid (, Graphical abstract Image 1

Surface Plasmon Resonance (SPR) is widely used in biological and chemical sensing with fascinating properties. However, the application of SPR to detect trace targets is hampered by non-specific binding and poor signal. A variety of approaches for amplification have been explored to overcome this deficiency including DNA aptamers as versatile target detection tools. Hybridization chain reaction (HCR) is a high-efficiency enzyme-free DNA amplification method operated at room temperature, in which two stable species of DNA hairpins coexist in solution until the introduction of the initiator strand triggers a cascade of hybridization events. At an optimal salt condition, as the concentrations of H1 and H2 increased, the HCR signals were enhanced, leading to signal amplification reaching up to 6.5-fold of the detection measure at 30 min. This feature enables DNA to act as an amplifying transducer for biosensing applications to provide an enzyme-free alternative that can easily detect complex DNA sequences. Improvement of more diverse recognition events can be achieved by integrating HCR with a phase-sensitive SPR (pSPR)-tested aptamer stimulus. This work seeks to establish pSPR aptamer system for highly informative sensing by means of an amplification HCR. Thus, combining pSPR and HCR technologies provide an expandable platform for sensitive biosensing.

During vertebrate development, the presomitic mesoderm (PSM) periodically segments into somites, which will form the segmented vertebral column and associated muscle, connective tissue, and dermis. The periodicity of somitogenesis is regulated by a segmentation clock of oscillating Notch activity. Here, we examined mouse mutants lacking onlyFgf4orFgf8, which we previously demonstrated act redundantly to prevent PSM differentiation.Fgf8is not required for somitogenesis, butFgf4mutants display a range of vertebral defects. We analyzedFgf4mutants by quantifying mRNAs fluorescently labeled by hybridization chain reaction within Imaris-based volumetric tissue subsets. These data indicate that FGF4 maintainsHes7levels and normal oscillatory patterns. To support our hypothesis that FGF4 regulates somitogenesis throughHes7, we demonstrate genetic synergy betweenHes7andFgf4, but not withFgf8. Our data indicate thatFgf4is potentially important in a spectrum of human Segmentation Defects of the Vertebrae caused by defective Notch oscillations.

The dysregulation of the concentration of individual circulating microRNAs or small sets of them has been recognized as a marker of disease. For example, an increase of the concentration of circulating miR-17 has been linked to lung cancer and metastatic breast cancer, while its decrease has been found in multiple sclerosis and gastric cancer. Consequently, techniques for the fast, specific and simple quantitation of microRNAs are becoming crucial enablers of early diagnosis and therapeutic follow-up. DNA based biosensors can serve this purpose, overcoming some of the drawbacks of conventional lab-based techniques. Herein, we report a cost-effective, simple and robust biosensor based on localized surface plasmon resonance and hybridization chain reaction. Immobilized gold nanoparticles are used for the detection of miR-17. Specificity of the detection was achieved by the use of hairpin surface-tethered probes and the hybridization chain reaction was used to amplify the detection signal and thus extend the dynamic range of the quantitation. Less than 1 h is needed for the entire procedure that achieved a limit of detection of about 1 pM or 50 amol/measurement, well within the reported useful range for diagnostic applications. We suggest that this technology could be a promising substitute of traditional lab-based techniques for the detection and quantification of miRNAs after these are extracted from diagnostic specimens and their analysis is thus made possible., Highlights • Over- or underexpression of microRNAs can serve as biomarkers for diseases, such as cancer. • A miRNA biosensor based on localized plasmon resonance and enzyme-free amplification is here reported. • The biosensor can quantitate specific microRNA in 1 h and can be multiplexed. • The limit of detection and specificity of the biosensor is within a diagnostically-useful range.

The visualization of multiple gene expressions in well-preserved tissues is crucial for the elucidation of physiological and pathological processes. In situ hybridization chain reaction (HCR) is a method to visualize specific mRNAs in diverse organisms by applying a HCR that is an isothermal enzyme-free nucleotide polymerization method using hairpin DNAs. Although in situ HCR is a versatile method, this method is not widely used by researchers because of their higher cost than conventional in situ hybridization (ISH). Here, we redesigned hairpin DNAs so that their lengths were half the length of commonly used hairpin DNAs. We also optimized the conjugated fluorophores and linkers. Modified in situ HCR showed sufficient fluorescent signals to detect various mRNAs such as Penk, Oxtr, Vglut2, Drd1, Drd2, and Moxd1 in mouse neural tissues with a high signal-to-noise ratio. The sensitivity of modified in situ HCR in detecting the Oxtr mRNA was better than that of fluorescent ISH using tyramide signal amplification. Notably, the modified in situ HCR does not require proteinase K treatment so that it enables the preservation of morphological structures and antigenicity. The modified in situ HCR simultaneously detected the distributions of c-Fos immunoreactivity and Vglut2 mRNA, and detected multiple mRNAs with a high signal-noise ratio at subcellular resolution in mouse brains. These results suggest that the modified in situ HCR using short hairpin DNAs is cost-effective and useful for the visualization of multiple mRNAs and proteins.

Hydrogen sulfide (H2S) in joint fluid acts as a signal molecule to regulate joint inflammation. Direct detection of H2S in joint fluid is of great significance for the diagnosis and treatment of arthritis. However, due to the low volume of joint fluid and low H2S concentration, existing methods face the problem of the insufficient limit of detection. In this study, a highly sensitive biosensor was proposed by designing a primer probe and combining it with hybrid chain reaction (HCR) under the strong interaction between metal ions and H2S to achieve H2S detection. The primer probe containing multiple cytosine (C) sequences was fixed on a gold electrode, and the C–Ag–C hairpin structure was formed under the action of Ag+. In the presence of H2S, it can combine with Ag+ in the hairpin structure to form Ag2S, which leads to the opening of the hairpin structure and triggers the hybridization chain reaction (HCR) with another two hairpin structures (H1 and H2). A large number of double-stranded nucleic acid structures can be obtained on the electrode surface. Finally, Ru(phen)32+ can be embedded into the double chain structure to generate the electrochemiluminescence (ECL) signal. The linear response of the H2S biosensor ranged from 0.1000 to 1500 nM, and the limit of detection concentration of H2S was 0.0398 nM. The developed biosensor was successfully used to determine H2S in joint fluid.

International audience; This paper aims at presenting a new magnetic labelling approach based on in-situ hybridization for the specific capture of bacterial cells. This approach is inspired by HCR-FISH, which was recently proposed as a new sensitive method for the detection of environmental microorganisms using a combination of FISH (Fluorescent In Situ Hybridization) and HCR (Hybridization Chain Reaction). Here we propose to adapt the technique to allow grafting of superparamagnetic nanoparticles onto target bacteria and therefore to exploit magnetism instead of fluorescence for their subsequent isolation using a micro-magnet array. The feasibility of the approach was first assessed by isolating E. coli cells using a universal bacterial probe, Eub338. Then, specific targeting of Escherichia coli DH5a, Pseudomonas putida, and Acinetobacter sp. ADP1 were demonstrated using heterologous probes. For the purpose of comparison, both HCR-FISH and HCR-MISH experimental results are presented.

In order to combat the growing threat of global infectious diseases, there is the need for rapid diagnostic technologies that are sensitive and that can provide species specific information (as might be needed to direct therapy as resistant strains of microbes emerge). Here, we present a convenient, enzyme-free amplification mechanism for a rational hybridization chain reaction (HCR), which is implemented in a simple format for isothermal amplification and sensing, applied to the DNA-based diagnosis of Hepatitis B virus (HBV) in 54 patients. During the cycled amplification process, DNA monomers self-assemble in an organized and controllable way, only when a specific target HBV sequence is present. This mechanism is confirmed using super-resolution Stochastic Optical Reconstruction Microscopy (STORM). The enabled format is designed in a manner analogous to an enzyme-linked immunosorbent assay (ELISA), generating coloured products with distinct tonality and with a limit of detection of ca. 5 copies/reaction. This routine assay also showed excellent sensitivity (>97%) in clinical samples demonstrating the potential of this convenient, low cost, enzyme-free method for use in low resource settings.

The visualization of multiple gene expressions in well-preserved tissues is crucial for the elucidation of physiological and pathological processes. In situ hybridization chain reaction (HCR) is a method to visualize specific mRNAs in diverse organisms by applying a HCR that is an isothermal enzyme-free nucleotide polymerization method using hairpin DNAs. Although in situ HCR is a versatile method, this method is not widely used by researchers because of their higher cost than conventional in situ hybridization (ISH). Here, we redesigned hairpin DNAs so that their lengths were half the length of commonly used hairpin DNAs. We also optimized the conjugated fluorophores and linkers. Modified in situ HCR showed sufficient fluorescent signals to detect various mRNAs such as Penk, Oxtr, Vglut2, Drd1, Drd2, and Moxd1 in mouse neural tissues with a high signal-to-noise ratio. The sensitivity of modified in situ HCR in detecting the Oxtr mRNA was better than that of fluorescent ISH using tyramide signal amplification. Notably, the modified in situ HCR does not require proteinase K treatment so that it enables the preservation of morphological structures and antigenicity. The modified in situ HCR simultaneously detected the distributions of c-Fos immunoreactivity and Vglut2 mRNA, and detected multiple mRNAs with a high signal-noise ratio at subcellular resolution in mouse brains. These results suggest that the modified in situ HCR using short hairpin DNAs is cost-effective and useful for the visualization of multiple mRNAs and proteins.

Graphical abstract A highly sensitive SERS assay is proposed for detecting DENV gene fragment via a cascade signal amplification strategy of localized catalytic hairpin assembly and hybridization chain reaction., Highlights • A highly sensitive SERS assay of DENV gene is proposed via a cascade signal amplification strategy of LCHA and HCR. • The SERS assay possesses high sensitivity with a linear calibration curve from 1 fM to 10 nM and a LOD low to 0.49 fM. • The SERS assay can provide a reliable tool for early diagnosis of virus infections., Pathogenic viruses with worldwide distribution, high incidence and great harm are significantly and increasingly threatening human health. However, there is still lack of sufficient, highly sensitive and specific detection methods for on-time and early diagnosis of virus infection. In this work, taking dengue virus (DENV) as an example, a highly sensitive SERS assay of DENV gene was proposed via a cascade signal amplification strategy of localized catalytic hairpin assembly (LCHA) and hybridization chain reaction (HCR). The SERS assay was performed by two steps, i.e., the operation of cascade signal amplification strategy and the following SERS measurements by transferring the products on SERS-active AgNRs arrays. The sensitivity of the cascade signal amplification strategy is significantly amplified, which is 4.5 times that of individual CHA, and the signal-to-noise ratio is also improved to 5.4 relative to 1.8 of the CHA. The SERS sensing possesses a linear calibration curve from 1 fM to 10 nM with the limit of detection low to 0.49 fM, and has good specificity, uniformity and recovery, which indicates that the highly sensitive SERS assay provides an attractive tool for reliable, early diagnosis of DENV gene and is worth to be popularized in a wide detection of other viruses.

In this work, hybridization chain reactions (HCRs) toward Severe Acute Respiratory Syndrome Coronavirus 2 (SARS–CoV-2) nucleocapsid phosphoproteins gene loci and human RNase P are proposed to provide an isothermal amplification screening tool. The proposed chain reactions target the complementary DNA (cDNA) of SARS–CoV-2, with loci corresponding to gold-standard polymerase chain reaction (PCR) loci. Four hybridization chain reaction reactions are demonstrated herein, targeting N1/N2/N3 loci and human RNase P. The design of the hybridization chain reaction, herein, is assisted with an algorithm. The algorithm helps to search target sequences with low local secondary structure and high hybridization efficiency. The loop domain of the fuel hairpin molecule H1 and H2, which are the tunable segments in such reactions, are used as an optimization parameter to improve the hybridization efficiency of the chain reaction. The algorithm-derived HCR reactions were validated with gel electrophoresis. All proposed reactions exhibit a hybridization complex with a molecular mass >1.5k base pairs, which is clear evidence of chain reaction. The hybridization efficiency trend revealed by gel electrophoresis corresponds nicely to the simulated data from the algorithm. The HCR reactions and the corresponding algorithm serve as a basis to further SARS–CoV-2 sensing applications and facilitate better screening strategies for the prevention of on-going pandemics.

Recent study proves that the combination of loop mediated isothermal nucleic acid amplification (LAMP) with one-step strand displacement (OSD) is of great help to improve the sequence specificity during genetic detection. However, because OSD is incapable of signal amplification, the signal-to-noise ratio or the observable signal change may be usually not significant enough to satisfy practical usage. With the purpose to overcome this challenge, herein a more advanced and practical sensing principle is developed with the OSD replaced by an amplifiable nucleic acid circuit, hybridization chain reaction (HCR). The very contagious norovirus (NoV) was employed as the model target. Compared with LAMP-OSD, the LAMP-HCR can detect as few as 30 copies of NoV gene in 2% fecal samples with significantly enlarged signal change and signal-to-background ratio. Therefore, more reliable detection is achieved. Moreover, due to the high compatibility of HCR, the final LAMP-HCR products can be flexibly transduced into different types of readouts, including fluorescence, flow cytometer (FCM) and even a personal glucose meter (PGM). This further enlarges the operating environments for the detection from hospital labs, bedsides, to potential off-the-shelf devices in local pharmacies. Especially when using FCM or PGM, with the assistance of magnetic beads (MBs), the detection shows even higher tolerance capability to complicated biological matrices., Graphical abstract A versatile molecular diagnostic method is developed for sensitive norovirus (NoV) gene detection by coupling the advantages of loop-mediated isothermal amplification (LAMP) with enzyme-free signal amplification hybridization chain reaction (HCR) for robust signal readout via flow cytometer (FCM) and personal glucometer (PGM).Image 1, Highlights • Highly sensitive and selective detection method for norovirus gene. • Sensitivity down to 30 gene copies in 2% fecal samples. • Significantly enlarged signal change and signal-to-background ratio. • Higher tolerance capability to complicated biological matrices. • Exhibiting great potential to be used in the diagnosis of gastroenteritis in hospital or at home.

By taking inspiration from nature, where self-organization of biomolecular species into complex systems is finely controlled through different stimuli, we propose here a rational approach by which the assembly and disassembly of DNA-based concatemers can be controlled through pH changes. To do so we used the hybridization chain reaction (HCR), a process that, upon the addition of an initiator strand, allows to create DNA-based concatemers in a controlled fashion. We re-engineered the functional units of HCR through the addition of pH-dependent clamp-like triplex-forming domains that can either inhibit or activate the polymerization reaction at different pHs. This allows to finely regulate the HCR-induced assembly and disassembly of DNA concatemers at either basic or acidic pHs in a reversible way. The strategies we present here appear particularly promising as novel tools to achieve better spatiotemporal control of self-assembly processes of DNA-based nanostructures.

DNA biosensors could overcome some of the common drawbacks of lab-based techniques for nucleic acids detection for diagnostics purposes. One of the main impediments for such applications of DNA biosensors is their lack of sensitivity: this can prevent their full exploitation in the diagnostic analytical field. DNA nanotechnology could enhance DNA biosensors and let them perform at the required high sensitivity. Well-designed, programmable self-assembly reactions can be triggered by a specific nucleic acid target. The Hybridization Chain Reaction (HCR) is a self-assembly strategy in which the target nucleic acid sequence triggers the formation of long nicked double-stranded DNA nanostructures. This can be performed in solution or on a surface, and the process can be coupled to different signal transduction schemes. We here describe the methods to design and test HCR reactions for the detection of different nucleic acid targets in solution and the procedures to exploit this strategy on surfaces with an electrochemical biosensing platform.

Dynamic RNA nanotechnology based on programmable hybridization cascades with small conditional RNAs (scRNAs) offers a promising conceptual framework for engineering programmable conditional regulation in vivo. While single-base substitution (SBS) somatic mutations and single-nucleotide polymorphisms (SNPs) are important markers and drivers of disease, it is unclear whether synthetic RNA signal transducers are sufficiently programmable to accept a cognate RNA input while rejecting single-nucleotide sequence variants. Here, we explore the limits of scRNA programmability, demonstrating isothermal, enzyme-free genotyping of RNA SBS cancer markers and SNPs using scRNAs that execute a conditional hybridization cascade in the presence of a cognate RNA target. Kinetic discrimination can be engineered on a time scale of choice from minutes to days. To discriminate even the most challenging single-nucleotide sequence variants, including those that lead to nearly isoenergetic RNA wobble pairs, competitive inhibition with an unstructured scavenger strand or with other scRNAs provides a simple and effective principle for achieving exquisite sequence selectivity.

Single-cell analysis is a rapidly evolving approach to characterize genome-scale molecular information at the individual cell level. Development of single-cell technologies and computational methods has enabled systematic investigation of cellular heterogeneity in a wide range of tissues and cell populations, yielding fresh insights into the composition, dynamics, and regulatory mechanisms of cell states in development and disease. Despite substantial advances, significant challenges remain in the analysis, integration, and interpretation of single-cell omics data. Here, we discuss the state of the field and recent advances and look to future opportunities., Genome Biology, 18, ISSN:1474-760X

In situ hybridization (ISH) techniques allow for the study of the nucleic acid expression within whole biological samples. The quality of probes for ISH dictates how accurate and bright the signal is for the experiment; however, there is currently not a systematic way to determine what the best probe set would be. In response to this, we have developed a framework to optimize an ISH probe set to achieve the greatest signal-to-background ratio. As methods like ISH help obtain more information about biological processes, there is a growing desire to simultaneously analyze various targets within the same sample to examine these complex genetic interactions. To facilitate this, a novel amplification technique called hybridization chain reaction (HCR) has allowed for the in situ detection of multiple target mRNAs concurrently in zebrafish embryos. We have now expanded this technology further by adapting HCR amplification for ISH to other model organisms, particularly, whole mount Drosophila melanogaster embryos and formalin-fixed parafin-embedded human tissue sections. Beyond looking at mRNA, immunohistochemistry (IHC) provides another tool to understand biological systems by analyzing protein expression patterns. The ability to easily look at both mRNAs and proteins in the same sample offers significant advantages as each provides unique information, but current methods are technically difficult and labor intensive. In response, we have engineered a scheme to use HCR to amplify signal for IHC. We then used this advancement to develop a straightforward protocol using HCR amplification for simultaneous detection of multiple proteins and mRNAs with a high signal-to-background ratio.

Morphogenesis is a phenomenon of intricate balance and dynamic interplay between processes occurring at a wide range of scales (spatial, temporal and energetic). During development, a variety of physical mechanisms are employed by tissues to simultaneously pattern, move, and differentiate based on information exchange between constituent cells, perhaps more than at any other time during an organism's life. To fully understand such events, a combined theoretical and experimental framework is required to assist in deciphering the correlations at both structural and functional levels at scales that include the intracellular and tissue levels as well as organs and organ systems. Microscopy, especially diffraction-limited light microscopy, has emerged as a central tool to capture the spatio-temporal context of life processes. Imaging has the unique advantage of watching biological events as they unfold over time at single-cell resolution in the intact animal. In this work I present a range of problems in morphogenesis, each unique in its requirements for novel quantitative imaging both in terms of the technique and analysis. Understanding the molecular basis for a developmental process involves investigating how genes and their products- mRNA and proteins-function in the context of a cell. Structural information holds the key to insights into mechanisms and imaging fixed specimens paves the first step towards deciphering gene function. The work presented in this thesis starts with the demonstration that the fluorescent signal from the challenging environment of whole-mount imaging, obtained by in situ hybridization chain reaction (HCR), scales linearly with the number of copies of target mRNA to provide quantitative sub-cellular mapping of mRNA expression within intact vertebrate embryos. The work then progresses to address aspects of imaging live embryonic development in a number of species. While processes such as avian cartilage growth require high spatial resolution and lower time resolution, dynamic events during zebrafish somitogenesis require higher time resolution to capture the protein localization as the somites mature. The requirements on imaging are even more stringent in case of the embryonic zebrafish heart that beats with a frequency of ~ 2-2.5 Hz, thereby requiring very fast imaging techniques based on two-photon light sheet microscope to capture its dynamics. In each of the hitherto-mentioned cases, ranging from the level of molecules to organs, an imaging framework is developed, both in terms of technique and analysis to allow quantitative assessment of the process in vivo. Overall the work presented in this thesis combines new quantitative tools with novel microscopy for the precise understanding of processes in embryonic development.

Molecular techniques to detect organisms in aquatic ecosystems are being gradually considered as an attractive alternative to standard laboratory methods. They offer faster and more accurate means of detecting and monitoring species, with respect to their traditional homologues based on culture and microscopic counting. Molecular techniques are particularly attractive when multiple species need to be detected and/or are in very low abundance. This paper reviews molecular techniques based on whole cells, such as microscope-based enumeration and Fluorescence In-Situ Hybridization (FISH) and molecular cell-free formats, such as sandwich hybridization assay (SHA), biosensors, microarrays, quantitative polymerase chain reaction (qPCR) and real time PCR (RT-PCR). Those that combine one or several laboratory functions into a single integrated system (lab-on-a-chip) and techniques that generate a much higher throughput data, such as next-generation systems (NGS), were also reviewed. We also included some other approaches that enhance the performance of molecular techniques. For instance, nano-bioengineered probes and platforms, pre-concentration and magnetic separation systems, and solid-phase hybridization offer highly pre-concentration capabilities. Isothermal amplification and hybridization chain reaction (HCR) improve hybridization and amplification techniques. Finally, we presented a study case of field remote sensing of harmful algal blooms (HABs), the only example of real time monitoring, and close the discussion with future directions and concluding remarks.

Nucleic acids are most commonly associated with the genetic code, transcription and gene expression. Recently, interest has grown in engineering nucleic acids for biological applications such as controlling or detecting gene expression. The natural presence and functionality of nucleic acids within living organisms coupled with their thermodynamic properties of base-pairing make them ideal for interfacing (and possibly altering) biological systems. We use engineered small conditional RNA or DNA (scRNA, scDNA, respectively) molecules to control and detect gene expression. Three novel systems are presented: two for conditional down-regulation of gene expression via RNA interference (RNAi) and a third system for simultaneous sensitive detection of multiple RNAs using labeled scRNAs. RNAi is a powerful tool to study genetic circuits by knocking down a gene of interest. RNAi executes the logic: If gene Y is detected, silence gene Y. The fact that detection and silencing are restricted to the same gene means that RNAi is constitutively on. This poses a significant limitation when spatiotemporal control is needed. In this work, we engineered small nucleic acid molecules that execute the logic: If mRNA X is detected, form a Dicer substrate that targets independent mRNA Y for silencing. This is a step towards implementing the logic of conditional RNAi: If gene X is detected, silence gene Y. We use scRNAs and scDNAs to engineer signal transduction cascades that produce an RNAi effector molecule in response to hybridization to a nucleic acid target X. The first mechanism is solely based on hybridization cascades and uses scRNAs to produce a double-stranded RNA (dsRNA) Dicer substrate against target gene Y. The second mechanism is based on hybridization of scDNAs to detect a nucleic acid target and produce a template for transcription of a short hairpin RNA (shRNA) Dicer substrate against target gene Y. Test-tube studies for both mechanisms demonstrate that the output Dicer substrate is produced predominantly in the presence of a correct input target and is cleaved by Dicer to produce a small interfering RNA (siRNA). Both output products can lead to gene knockdown in tissue culture. To date, signal transduction is not observed in cells; possible reasons are explored. Signal transduction cascades are composed of multiple scRNAs (or scDNAs). The need to study multiple molecules simultaneously has motivated the development of a highly sensitive method for multiplexed northern blots. The core technology of our system is the utilization of a hybridization chain reaction (HCR) of scRNAs as the detection signal for a northern blot. To achieve multiplexing (simultaneous detection of multiple genes), we use fluorescently tagged scRNAs. Moreover, by using radioactive labeling of scRNAs, the system exhibits a five-fold increase, compared to the literature, in detection sensitivity. Sensitive multiplexed northern blot detection provides an avenue for exploring the fate of scRNAs and scDNAs in tissue culture.

With the goal of determining the presence of pathogens when present in very small amount, such as for the screening of pathogens in drinking water, an analytical method of very high sensitivity must be implemented. As often this type of determinations should be performed at the point-of-care, with simple, automatic and inexpensive hardware, a chemical (or nanotechnological) signal-amplification step is required prior to biosensor read-out. The hybridization chain reaction (HCR), originally proposed by Dirks and Pierce (2004), is an isothermal, enzyme-free process where a hybridization event triggers the polymerization of oligonucleotides into a long albeit nicked double-stranded nucleic acid molecule. The two kinds of co-monomer oligonucleotides are present in solution as closed hairpins, are opened and added to the growing polymer only after the initial triggering hybridization event. We have designed HCR oligonucleotide sets to target DNA sequences of relevance for the protection from bioterrorism acts towards water systems. In this communication, we presents our results of the HCR amplifications for different DNA targets of C. parvum, G. lamblia and Hepatitis E virus that have been performed both in solution and in a microarray format, to make it more amenable of use in diagnostic or point-of-interest assays. Our need of detecting natural occurring sequences forcedly limits our possibility of sequence optimization, reducing it to the selection of a target sequence with low secondary structure within a consensus sequence defined by bioinformatics tools. The design of the HCR hairpins was the performed on the defined target sequences with the aid of NANEV (Goodman, 2005). Our results seem to point out that there is a general sensitivity advantage in a biosensor read-out of the presence of naturally occurring nucleic acids sequences when the hybridization chain reaction is used as a signal-amplification step. This strategy could result of general interest for the analytical determination of nucleic acids.

INTRODUCTION: Single-molecule sensitivity can be nowadays achieved when trying to characterize minute amounts of macromolecules n in a research laboratory setting. The sensitivity is significantly worse when working in the field and trying to assess the presence of a pollutant or a pathogen as quickly as possible. Point-of-care testing is the realm of biosensors and the trend of research is towards simplicity of use, low cost, reliability and, last but not least, sensitivity. In the detection of nucleic acids, the hybridization of an oligonucleotide probe with its poly-nucleotidic target is monitored by changes in physico-chemical properties of an interface, commonly induced by the use of a specific label. The probe, often bound to an oligonucleotide itself, can be a fluorescent dye, an enzyme, an electroactive moiety or other functional element. Such probes impart specificity and sensitivity to the assay, but make it drift far from simplicity of use (and low cost). METHODS AND RESULTS: Recently, label-free techniques are developing in which the presence of the analyte macromolecule itself can induce detectable physico-chemical changes. The struggle is to make such techniques sufficiently sensitive and specific. In our work, we are trying to implement DNA-based surface-bound amplification strategies that can serve to amplify the read-out signal of DNA hybridization in a label-free biosensor, such as those measuring the electrochemical properties of an electrode interface. The ‘hybridization chain reaction’ [1] is an isothermal enzyme-free strategy to trigger polymerization of oligonucleotides into a long double-stranded DNA. We have demonstrated that the ‘hybridization chain reaction’ can be also implemented (making use of available software tools [2]) in a surface-bound configuration, leading to the self-assembly of many copies of oligonucleotides on a target DNA bound on an oligonucleotide self-assembled monolayer. In another attempt, we have shown that terminal transferase (a template-free DNA polymerase) can be used to build a long polynucleotide out of the target DNA that is bound to its immobilized oligonucleotide probe. The Rolling Circle Amplification reaction is a very promising strategy to produce large amounts of DNA without the need of thermal cycling or of an accurate control of temperature. Preliminary evaluations on the Primer-Generation RCA [3] show its interest as an additional strategy to increase the amount of target DNA response from a biosensor surface. DISCUSSION & CONCLUSIONS: All these strategies will lead to the accumulation of nucleic acids at the solid-liquid interface when triggered by the probe-target recognition. This produces an amplification of the signal of a label-free biosensor. Even though the amplification factors of these implementations are still as low as 10, their further development is still possible, with the hope of avoiding or reducing the need of polymerase chain reaction in the detection of low concentrations of nucleic acids. REFERENCES: 1Dirks, R. M. and N. A. Pierce (2004). "Triggered amplification by hybridization chain reaction." Proc Natl Acad Sci U S A, 101(43): 15275-8. 2Goodman, P. (2005). "NANEV: a program employing evolutionary methods for the design of nucleic acid nanostructures" BioTechniques, 38(4): 548–550. 3Murakami, Sumaoka and Komiyama (2009) "Sensitive isothermal detection of nucleic-acid sequence by primer generation–rolling circle amplification" Nucleic Acids Research, 2009, 37, e19. ACKNOWLEDGEMENTS: This work was supported by Framework Programme 6 Integrated Project DINAMICS.

In situ hybridization methods enable the mapping of mRNA expression within intact biological samples. With current approaches, it is challenging to simultaneously detect multiple target mRNAs in vertebrate embryos and tissue sections – a significant limitation in attempting to study interacting regulatory elements in systems most relevant to human development and disease. This thesis presents a multiplexed fluorescent in situ hybridization method based on orthogonal amplification with hybridization chain reaction (HCR). Using this approach, RNA probes complementary to mRNA targets trigger chain reactions in which fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. Robust performance and high signal-to-background are achieved when imaging five target mRNAs at the same time in fixed whole-mount zebrafish embryos. The programmability and sequence specificity of these amplification cascades enable all five amplifiers to operate orthogonally at the same time in the same sample. The fact that amplification polymers are triggered to self-assemble in situ results in excellent sample penetration and high signal-to-background. These properties suggest the broad applicability of fluorescent in situ HCR amplification to multiplexed imaging of mRNA expression in normal and pathological cells, embryos, and tissue sections.

The hybridization chain reaction (HCR), in which stable DNA monomers assemble only upon exposure to a target DNA fragment, has been introduced earlier for reactions in solution. Two species of DNA hairpins are present in solution without interaction, until an initiator DNA triggers a cascade of hybridization events resulting in an elongated strand of alternating opened hairpins forming a double helix. Hybridization chain reaction (HCR) was introduced as an attractive method to amplify the signal of targeted nucleic acids. We report here the results of the HCR for the amplification of DNA microarray signals. Hairpins were fluorescently labelled with Cy3 or Cy5, or with alternative couples of fluorophors. First results indicate that the HCR-induced fluorescence at the surface might be hard to detect directly. Protocols involving the removal of the amplification product from the surface evidence an amplification factor of about 5. Performing the HCR in solution before hybridizing the product to the DNA microarray, i.e. using HCR for multiple labelling the target for the detection, produces a signal amplification of a factor 3-4. The effect of buffer composition, initiator concentration and hairpin concentration was investigated. The low sensitivity that is currently attainable (approximately 0,05 µM) severely limits the practical use of the HCR.

Nucleic acids present great promise as building blocks for nanoscale devices. To achieve this potential, methods for the analysis and design of DNA and RNA need to be improved. In this thesis, traditional algorithms for analyzing nucleic acids at equilibrium are extended to handle a class of pseudoknots, with examples provided relevant to biologists and bioengineers. With these analytical tools in hand, nucleic acid sequences are designed to maximize the equilibrium probability of a desired fold. Upon analysis, it is concluded that both affinity and specificity are important when choosing a sequence; this conclusion holds for a wide range of target structures and is robust to random perturbations to the energy model. Applying the intuition gained from these studies, a process called hybridization chain reaction (HCR) is invented, and sequences are chosen that experimentally verify this phenomenon. In HCR, a small number of DNA or RNA molecules trigger a system wide configurational change, allowing the amplification and detection of specific, nucleic acid sequences. As an extension, HCR is combined with a pre-existing aptamer domain to successfully construct an ATP sensor, and the groundwork is laid for the future development of sensors for other small molecules. In addition, recent studies on multi-stranded algorithms and improvements to HCR are included in the appendices. Not only will these advancements increase our understanding of biological RNAs, but they will also provide valuable tools for the future development of nucleic acid nanotechnologies.

A more specific and intense signal is desirable for most kinds of biosensors for biomedical or environmental applications, and it is especially so for label-free biosensors. In this paper, we show that hybridization chain reaction (HCR) can be exploited for the easily detectable accumulation of nucleic acids on metal surfaces as an event triggered by specific recognition between a probe and a target nucleic acid. We show that this process could be exploited to increase the sensitivity in the detection of nucleic acids derived from a pathogenic microorganism. This strategy can be straightforwardly implemented on SPR biosensors (commercial or custom-built) or on label-free electrochemical biosensors. Together with signal amplification, HCR can serve as a confirmation of the specificity of target recognition, as it involves the specific matching with a separate base sequence in the target nucleic acid. Furthermore, the kinetics of the target binding and the HCR can be easily distinguished from each other, providing an additional means of confirmation of the specific recognition. (C) 2013 Elsevier B.V. All rights reserved.