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Despite their unique optical and electrical characteristics, traditional semiconductor quantum dots (QDs) made of heavy metals or carbon are not compatible with many biomedical applications. Cytotoxicity and environmental concerns are key limiting factors that prevent their widescale transition from laboratory research to real-world medical applications. Recently, advanced InP/ZnSe/ZnS QDs have emerged as excellent alternatives to traditional QDs due to their lower toxicity and optical properties; however, they fall short of traditional QDs with respect to their versatility for bioconjugation (i.e., surface chemistry limitations causing unstability in aqueous environments). In this work, we construct a road map for generating, for the first time, highly efficient bio-templated InP/ZnSe/ZnS-aptamers (QDAPT) with long-term stability and high selectivity for applications in targetting bacterial membrane proteins. Our QDAPTs show fast binding reaction kinetics (less than 5 minutes), high brightness, and high shelf-life stability (3 months) after biotemplation in aqueous solvents. We also demonstrate the detection of bacterial membrane proteins on common surfaces using a hand-held imaging device, which attests to the great potential of this system for incorporation into future biomedical technologies.

There is a constant drive for affordable point-of-care testing (POCT) technologies for the detection of infectious human diseases. Herein, we report a simple platform for DNA detection that takes advantage of four techniques: commercially available pregnancy test strips (PTS), amplicon generation via loop-mediated isothermal amplification (LAMP), toehold-mediated strand displacement, and noncovalent immobilization of DNA on paper surface with DNA nanoflowers. This simple, separation-free platform is highly specific, as demonstrated with the detection of rtL180M, a single-nucleotide polymorphism observed in hepatitis B virus (HBV) associated with antiviral drug resistance. It is very sensitive, capable of detecting the targeted mutation at 2 copies μL-1 . It is able to correctly identify the unmutated and rtL180M genome types of HBV in clinical samples. Given its wide adaptability, we expect this platform can be easily modified for the detection of genetic variations associated with various pathogens and human diseases.

ConspectusPathogens have long presented a significant threat to human lives, and hence the rapid detection of infectious pathogens is vital for improving human health. Current detection methods lack the means to detect infectious pathogens in a simple, rapid, and reliable manner at the time and point of need. Functional nucleic acids (FNAs) have the potential to overcome these limitations by acting as key components for point-of-care (POC) biosensors due to their distinctive advantages that include high binding affinities and specificities, excellent chemical stability, ease of synthesis and modification, and compatibility with a variety of signal-amplification and signal-transduction mechanisms.This Account summarizes the work completed in our groups toward developing FNA-based biosensors for detecting bacteria. In vitro selection has led to the isolation of many RNA-cleaving fluorogenic DNAzymes (RFDs) and DNA aptamers that can recognize infectious pathogens, including Escherichia coli, Clostridium difficile, Helicobacter pylori, and Legionella pneumophila. In most cases, a "many-against-many" approach was employed using a DNA library against a crude cellular mixture of an infectious pathogen containing diverse biomarkers as the target to isolate RFDs, with combined counter and positive selections ensuring high specificity toward the desired target. This procedure allows for the isolation of pathogen-specific FNAs without first identifying a suitable biomarker. Multiple target-specific DNA aptamers, including anti-glutamate dehydrogenase (GDH) circular aptamers, anti-degraded toxin B aptamers, and anti-RNase HII aptamers, have also been isolated for the detection of bacteria such as Clostridium difficile. The isolated FNAs have been integrated into fluorescent, colorimetric, and electrochemical biosensors using various signal transduction mechanisms. Both simple-to-use paper-based analytical devices and hand-held electrical devices with integrated FNAs have been developed for POC applications. In addition, signal-amplification strategies, including DNA catenane enabled rolling circle amplification (RCA), DNAzyme feedback RCA, and an all-DNA amplification system using a four-way junction and catalytic hairpin assembly (CHA), have been designed and applied to these systems to further increase their detection sensitivity. The use of these FNA-based biosensors to detect pathogens directly in clinical samples, such as urine, blood, and stool, has now been demonstrated with an outstanding sensitivity of as low as 10 cells per milliliter, highlighting the tremendous potential of using FNA-based sensors in clinical applications. We further describe strategies to overcome the challenges of using FNA-based biosensors in clinical applications, including strategies to improve the stability of FNAs in biological samples and prevent their nonspecific degradation from nucleases and strategies to deal with issues such as signal loss caused by nonspecific binding and biofouling. Finally, the remaining roadblocks for employing FNA-based biosensors in clinical applications are discussed.

Adenosine 5'-triphosphate (ATP) is a central extracellular signaling agent involved in various physiological and pathological processes. However, precise measurements of the temporal and spatial components of ATP dynamics are lacking due primarily to the limitations of available methods for ATP detection. Here, we report on the first effort to design a self-phosphorylating DNAzyme (SPDz) sensor for fluorescence imaging of ATP. In response to ATP, SPDz sensors exhibit subsecond response kinetics, extremely high specificity, and micromolar affinities. In particular, we demonstrate cell-surface-anchored SPDz sensors for fluorescence imaging of both stress-induced endogenous ATP release in astrocytes and mechanical stimulation-evoked ATP release at the single-cell level. We also validated their utility for visualizing the rapid dynamic properties of ATP signaling upon electrical stimulation in astrocytes. Thus, SPDz sensors are robust tools for monitoring ATP signaling underlying diverse cellular processes.

Pen-side testing of farm animals for infectious diseases is critical for preventing transmission in herds and providing timely intervention. However, most existing pathogen tests have to be conducted in centralized labs with sample-to-result times of 2-4 days. Herein we introduce a test that uses a dual-electrode electrochemical chip (DEE-Chip) and a barcode-releasing electroactive aptamer for rapid on-farm detection of porcine epidemic diarrhea viruses (PEDv). The sensor exploits inter-electrode spacing reduction and active field mediated transport to accelerate barcode movement from electroactive aptamers to the detection electrode, thus expediting assay operation. The test yielded a clinically relevant limit-of-detection of 6 nM (0.37 μg mL

Molecular recognition elements with high specificity are of great importance for the study of molecular interactions, accurate diagnostics, drug design, and personalized medicine. Herein, a highly specific DNA aptamer for RNase H2 from Clostridium difficile (C. difficile) was generated by SELEX and minimized to 40 nucleotides. The aptamer exhibits a dissociation constant (Kd) of 1.8 ± 0.5 nM and an inhibition constant (IC50) of 7.1 ± 0.6 nM for C. difficile RNase H2, both of which are 2 orders of magnitude better for the same enzyme from other control bacteria. The fluorescent version of the aptamer can distinguish C. difficile from several other control bacteria in a cell lysate assay. This work demonstrates that a ubiquitous protein like RNase H2 can still be used as the target for the development of highly specific aptamers and the combination of the protein and the aptamer can achieve the recognition specificity needed for a diagnostic test and drug development.

Circular DNA aptamers are powerful candidates for therapeutic applications given their dramatically enhanced biostability. Herein we report the first effort to evolve circular DNA aptamers that bind a human protein directly in serum, a complex biofluid. Targeting human thrombin, this strategy has led to the discovery of a circular aptamer, named CTBA4T-B1, that exhibits very high binding affinity (with a dissociation constant of 19 pM), excellent anticoagulation activity (with the half maximal inhibitory concentration of 90 pM) and high stability (with a half-life of 8 h) in human serum, highlighting the advantage of performing aptamer selection directly in the environment where the application is intended. CTBA4T-B1 is predicted to adopt a unique structural fold with a central two-tiered guanine quadruplex capped by two long stem–loops. This structural arrangement differs from all known thrombin binding linear DNA aptamers, demonstrating the added advantage of evolving aptamers from circular DNA libraries. The method described here permits the derivation of circular DNA aptamers directly in biological fluids and could potentially be adapted to generate other types of aptamers for therapeutic applications.

Protein biomarkers often exist as degradation fragments in biological samples, and affinity agents derived using a purified protein may not recognize them, limiting their value for clinical diagnosis. Herein, we present a method to overcome this issue, by selecting aptamers against a degraded form of the toxin B protein, which is a marker for diagnosing toxigenic Clostridium difficile infections. This approach has led to isolation of a DNA aptamer that recognizes degraded toxin B, fresh toxin B, and toxin B spiked into human stool samples. DNA aptamers selected using intact recombinant toxin B failed to recognize degraded toxin B, which is the form present in stored stool samples. Using this new aptamer, we produced a simple paper-based analytical device for colorimetric detection of toxin B in stool samples, or in the NAP1 strain of Clostridium difficile. The combined aptamer-selection and paper-sensing strategy can expand the practical utility of DNA aptamers in clinical diagnosis.

The engineering of easy-to-use biosensors with ultra-low detection sensitivity remains a major challenge. Herein, we report a simple approach for creating such sensors through the use of an RNA-cleaving DNAzyme (RcD) and a strategy designed to concentrate its cleavage product significantly. The assay uses micron-sized beads loaded with a target-responsive RcD and a paper strip containing a microzone covered with a DNA oligonucleotide capable of capturing the cleavage product of the RcD through Watson-Crick hybridization. Placing the beads and the paper strip in a target-containing test sample allows the bead-bound RcD molecules to undergo target-induced RNA cleavage, releasing a DNA fragment that is captured by the paper strip. This strategy, though simple, is very effective in achieving high levels of detection sensitivity, being able to enrich the concentration of the cleavage product by three orders of magnitude. It is also compatible with both fluorescence-based and colorimetric reporting mechanisms. This work provides a simple platform for developing ultrasensitive biosensors that take advantage of the widely available RcDs as molecular recognition elements.

Ribonuclease I belongs to a class of nonspecific endoribonucleases and plays many important roles in a variety of biological and cellular processes. While their ubiquitous nature and high activity contribute to the well-known problem of RNase contamination in experimentation, their abundance in bacteria can potentially be leveraged as a biosensor target. As a result, there is substantial interest in generating a specific and reliable probe for RNase detection for a variety of purposes. To that end, we report on our unintentional discovery of the RNase I probe RFA13-1 isolated through in vitro selection with the crude extracellular mixture from Clostridium difficile contaminated with Klebsiella aerogenes as a selection target. Characterization of RFA13-1 reveals that it exhibits high sensitivity to Escherichia coli RNase I with a detection limit of 1.39 pm. Furthermore, RFA13-1 also shows high specificity for RNase I produced only in select bacteria from the Enterobacteriaceae family. As a result, this probe offers a simple tool for RNase I detection with potential applications in RNase functional studies, ribonuclease contamination monitoring, and bacterial detection.

The development of new methods for direct viral detection using streamlined and ideally reagent-free assays is a timely and important, but challenging, problem. The challenge of combatting the COVID-19 pandemic has been exacerbated by the lack of rapid and effective methods to identify viral pathogens like SARS-CoV-2 on-demand. Existing gold standard nucleic acid-based approaches require enzymatic amplification to achieve clinically relevant levels of sensitivity and are not typically used outside of a laboratory setting. Here, we report reagent-free viral sensing that directly reads out the presence of viral particles in 5 minutes using only a sensor-modified electrode chip. The approach relies on a class of electrode-tethered sensors bearing an analyte-binding antibody displayed on a negatively charged DNA linker that also features a tethered redox probe. When a positive potential is applied, the sensor is transported to the electrode surface. Using chronoamperometry, the presence of viral particles and proteins can be detected as these species increase the hydrodynamic drag on the sensor. This report is the first virus-detecting assay that uses the kinetic response of a probe/virus complex to analyze the complexation state of the antibody. We demonstrate the performance of this sensing approach as a means to detect, within 5 min, the presence of the SARS-CoV-2 virus and its associated spike protein in test samples and in unprocessed patient saliva.

Molecular recognition elements with high specificity are of great importance for the study of molecular interactions, accurate diagnostics, drug design, and personalized medicine. Herein, a highly specific DNA aptamer for RNase H2 from

Aptamers are molecular recognition elements made of nucleic acids. Diverse synthetic aptamers have been discovered in a large number of SELEX experiments since 1990. This review begins with the analysis of these SELEX experiments by examining the range of targets as well as the affinity and specificity for these targets by DNA, RNA or modified nucleic acid aptamers generated from these experiments. This is followed by comparisons of synthetic aptamers with natural RNA aptamers from riboswitches and with some of the best naturally occurring protein based recognition elements for proteins and small molecules. These comparisons reveal the gaps between man-made aptamers and natural recognition elements. We then put forward a series of ideas for consideration by the aptamer community towards developing better aptamers to solve real world problems. These include performing aptamer selections with libraries containing larger random sequence domains to derive larger aptamers with more intricate structures, conducting more reselection experiments using mutagenized DNA libraries based on initially selected aptamer sequences to search for better members of an aptamer family, selecting aptamers that are programmed to recognize two or more different epitopes of the same target in order to build multivalent aptamers for increased affinity, expanding the effort of selecting aptamers using modified nucleic acids with enhanced chemical functionalities, innovating SELEX methods to drive for the selection of aptamers with truly outstanding affinity and specificity, and having terminal applications in mind when creating new aptamers so that they are highly functional in the environment where the applications are planned.

Herein, we report on the design of a programmable DNA ribbon using long-chain DNA molecules with a user-defined repetitive padlock sequence. The DNA ribbon can be further combined with gold nanoparticles (AuNPs) to create a composite nanomaterial that contains an AuNP core and a high-density DNA crown carrying a cancer-cell-targeting DNA aptamer, a fluorescent tag for location tracking, and a cell-killing drug. This composite material can be efficiently internalized by cancer cells and its cellular location can be tracked by fluorescence imaging. The system offers several attractive characteristics, including simple design, tunable DNA crown, high drug-loading capacity, selective cell targeting, and pH-sensitive drug release. These features make such a material a promising therapeutic agent.

The detection and identification of bacteria currently rely on enrichment steps such as bacterial culture and nucleic acid amplification to increase the concentration of target analytes. These steps increase assay time, cost and complexity, making it difficult to realize a truly rapid point-of-care test. Here we report the development of an electrical assay that uses electroactive RNA-cleaving DNAzymes (e-RCDs) to identify specific bacterial targets and subsequently release a DNA barcode for transducing a signal onto an electrical chip. Integrating e-RCDs into a two-channel electrical chip with nanostructured electrodes provides the analytical sensitivity and specificity needed for clinical analysis. The e-RCD assay is capable of detecting 10 CFU (equivalent to 1,000 CFU ml–1) of Escherichia coli selectively from a panel containing multiple non-specific bacterial species. Clinical evaluation of this assay using 41 patient urine samples demonstrated a diagnostic sensitivity of 100% and specificity of 78% at an analysis time of less than one hour compared with the several hours needed for currently used culture-based methods. Methods to detect and identify bacteria typically rely on enrichment steps such as bacterial culture and nucleic acid amplification. Now, an assay for detecting bacteria based on a two-channel electrical chip that combines electroactive DNAzymes with an electrochemical readout, has been developed. This assay enables reagentless and culture-free detection of bacteria in clinical samples.

DNA is a highly programmable material that can be configured into unique high-order structures, such as DNA branched junctions containing multiple helical arms converging at a center. Herein we show that DNA programmability can deliver in situ growth of a 3-way junction-based DNA structure (denoted Y-shaped DNA) with the use of three hairpin-shaped DNA molecules as precursors, a specific microRNA target as a recyclable trigger, and a DNA polymerase as a driver. We demonstrate that the Y-shaped configuration comes with the benefit of restricted freedom of movement in confined cellular environment, which makes the approach ideally suited for in situ imaging of small RNA targets, such as microRNAs. Comparative analysis illustrates that the proposed imaging technique is superior to both the classic fluorescence in situ hybridization (FISH) method and an analogous amplified imaging method via programmed growth of a double-stranded DNA (rather than Y-shaped DNA) product.

DNA-based enzymes, or DNAzymes, are not known to exist in Nature but can be isolated from random-sequence DNA pools using test tube selection techniques. Since the report of the first DNAzyme in 1994, many catalytic DNA molecules for catalyzing wide-ranging chemical transformations have been isolated and studied. Our laboratory has a keen interest in searching for diverse DNAzymes capable of cleaving RNA-containing substrates, determining their sequence requirements and structural properties, and examining their potential as biosensors. This Account begins with the description of an accidental discovery on the sequence adaptability of a small DNAzyme known as "8-17", when we performed 16 parallel selections to search for DNAzymes that targeted each and every possible dinucleotide junction of RNA for cleavage. DNAzyme 8-17 dominated all the selection pools targeting purine-containing junctions. In-depth sequence analysis revealed that 8-17 could manifest itself in many sequence options defined by the requirement of four absolutely conserved nucleotides. This study also exposed the fact that 8-17 had poor activity toward pyrimidine-pyrimidine junctions. With this information in hand, we proceeded to the discovery of diverse non-8-17 DNAzymes that exhibited robust catalytic activity under physiological conditions. These DNAzymes were found to universally interact with their substrates through two Watson-Crick binding arms and have a catalytic core of varying length and secondary-structure complexity. RNA-cleaving DNAzymes were also isolated to function at acidic conditions (pH 3-5), and these molecules exhibited intriguing pH profiles, with the highest activity precisely matching the pH used for their selection. Interestingly, these DNAzymes appear to use non-Watson-Crick interactions in defining their structures. More recently, we have embarked on the development of ligand-responsive RNA-cleaving fluorogenic DNAzymes that can recognize specific bacterial pathogens, such as Escherichia coli and Clostridium difficile, using a method that does not require a priori identification of a specific biomarker. Instead, the crude extracellular mixture as a whole is used as the target to drive the DNAzyme isolation. High recognition specificity can be achieved with a double-selection approach in which a DNA library is negatively selected against the cellular mixture prepared from unintended bacteria, followed by positive selection against the same mixture derived from a specific species or strain of bacterial pathogen. Finally, we have shown that DNAzymes' compatibility with DNA replication can benefit the design of amplification mechanisms that uniquely link the action of RNA-cleaving DNAzymes to rolling circle amplification, an isothermal DNA amplification technique. These methods are well suited for translating the target-binding and cleavage activity of an analyte-activated RNA-cleaving DNAzyme into the production of massive amounts of DNA amplicons to achieve ultrahigh detection sensitivity. Given the high chemical stability of DNA, our ability to discover catalytic DNA sequences by simultaneously evaluating as many as 10

Single-stranded DNA molecules capable of molecular recognition and catalysis can now be routinely generated via the technique of in vitro selection. When coupled with adequate signal transduction modes, these synthetic functional DNA species represent a potential paradigm shift in the research and development of biosensors to meet the challenges of our rapidly changing world. Coupling functional DNA molecules with graphene materials for the design of optical biosensors has become an exciting research area in recent years, mostly because graphene materials are not only excellent quenchers of fluorescence, but they also display considerably different affinities for free and ligand-bound functional DNA molecules. We will discuss notable progress in this area in this mini-review by highlighting representative studies.

Pathogenic strains of bacteria are known to cause various infectious diseases and there is a growing demand for molecular probes that can selectively recognize them. Here we report a special DNAzyme (catalytic DNA), RFD‐CD1, that shows exquisite specificity for a pathogenic strain of Clostridium difficile (C. difficile). RFD‐CD1 was derived by an in vitro selection approach where a random‐sequence DNA library was allowed to react with an unpurified molecular mixture derived from this strain of C. difficle, coupled with a subtractive selection strategy to eliminate cross‐reactivities to unintended C. difficile strains and other bacteria species. RFD‐CD1 is activated by a truncated version of TcdC, a transcription factor, that is unique to the targeted strain of C. difficle. Our study demonstrates for the first time that in vitro selection offers an effective approach for deriving functional nucleic acid probes that are capable of achieving strain‐specific recognition of bacterial pathogens.

Integrating functional DNA probes with graphene oxide (GO) has proved to be a productive approach to the design of optical biosensors that detect nucleic acids, proteins or small-molecule targets. This distinctive methodology neatly combines four intriguing properties associated with DNA and GO: • the functional versatility of DNA sequences as molecular receptors; • the ability of DNA to switch structural forms in a target-responsive manner; • the power of GO as a fluorescence quencher; and, • the differential affinity of GO for different structural forms of DNA. In recent years, several elegant signal-amplification strategies uniquely applicable to optical DNA-GO biosensors were reported and these amplified biosensors were productively employed to achieve ultrasensitive detection of diverse targets. This review highlights notable achievements in this budding research field and provides a perspective on where the field is heading.

Urinary tract infections (UTIs) are one of the most ubiquitous bacterial infection diseases affecting half of the global population at least once in their lifetime. Given the occurrence of the false positive result in Dipstics and the time-consuming identification of bacteria using culturing method to detect UroPathogenic Escherichia coli (UPEC), there is the need for a point-of-care diagnostic platform that is reliable, sensitive, specific, cost-effective, fast and easy to use. With the advent electrochemical methods coupled with the specific biorecognition elements, the detection of any biomarker has become possible with one touch. Among many biorecognition molecules, DNAzymes are gaining attention due to their robustness, stability, specificity, and the ease of selection without prior knowledge of the biomarker. Their structure switching and functionality (RNA cleaving) can be utilized with redox active species (like methylene blue) to design highly sensitive and selective electrochemical assays for pathogen detection. In this work, we have developed a dual signaling electrochemical DNAzyme based platform for specific and sensitive detection of Escherichia coli (E.coli) in UTI patient urine samples. The two on-chip working electrodes named as release channel and capture channel are integrated with DNAzyme-redox barcode and DNA probe respectively. In the presence of the bacterial target the DNAzyme catalyzes the cleavage of the redox DNA barcode in the release channel, which is subsequently translated into a redox signal upon hybridization with the probe on the capture channel. The differential signal generated by the two channels in the sensor demonstrated the sensitive detection of 10 CFU for E.coli in buffer and urine. Additionally, the sensor specifically detected E. coli in the presence of a panel of other gram positive and negative bacteria. Clinical validation of the assay was performed using 40 E.coli+ and E.coli- patient urine delineating a clinical sensitivity of 100% and specificity of 78% in a 30 minute testing time. This cost effective, reagent-free, time-efficient, and amplification-free electrochemical assay is suitable for detecting a wide range of pathogens, also alleviates the additional knowledge requirement of the biomarker as compared to other protein and aptamer-based assay. With the advancement in the chip fabrication and microfluidics, this assay can also be multiplexed with other pathogen for simultaneous detection of multiple pathogens in a single urine sample.

We report a signal amplification strategy termed DNAzyme feedback amplification (DFA) that takes advantage of rolling-circle amplification (RCA) and an RNA-cleaving DNAzyme (RCD). DFA employs two specially programmed DNA complexes, one composed of a primer and a circular template containing the antisense sequence of an RCD, and the other composed of the same circular template and an RNA-containing substrate for the RCD. RCA is initiated at the first complex to produce RCD elements that go on to cleave the substrate in the second complex. This cleavage event triggers the production of more input complexes for RCA. This reaction circuit continues autonomously, resulting in exponential DNA amplification. We demonstrate the versatility of this approach for biosensing through the design of DFA systems capable of detecting a microRNA sequence and a bacterium, with sensitivity improvements of 3-6 orders of magnitude over conventional methods.

Biosensors are analytical devices that have found a variety of applications in medical diagnostics, food quality control, environmental monitoring and biodefense. In recent years, functional nucleic acids, such as aptamers and nucleic acid enzymes, have shown great potential in biosensor development due to their excellent ability in target recognition and catalysis. Deoxyribozymes (or DNAzymes) are single-stranded DNA molecules with catalytic activity and can be isolated to recognize a wide range of analytes through the process of in vitro selection. By using various signal transduction mechanisms, DNAzymes can be engineered into fluorescent, colorimetric, electrochemical and chemiluminescent biosensors. Among them, colorimetric sensors represent an attractive option as the signal can be easily detected by the naked eye. This reduces reliance on complex and expensive equipment. In this review, we will discuss the recent progress in the development of colorimetric biosensors that make use of DNAzymes and the prospect of employing these sensors in a range of chemical and biological applications.

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

DNAzymes refer to single-stranded DNA molecules with catalytic activity and can be isolated from synthetic random-sequence DNA pools using the technique of in vitro selection. DNAzymes that cleave RNA, known as "RNA-cleaving DNAzymes", represent one of the best-studied classes of DNAzymes and have been widely used for the development of biosensors and bioassays for various analytes. We have been interested in developing RNA-cleaving DNAzymes as bacterial sensors and these DNAzymes are engineered to perform three linked functions: recognition of a bacterial biomarker, RNA cleavage, and fluorescence generation. These fluorogenic DNAzymes emit fluorescence automatically in the presence of a bacterium of interest and can be used to set up a simple "mix-and-read" assay to detect this bacterium. In this article, we will discuss this DNAzyme system and present a proven strategy for isolating highly specific bacteria-responding DNAzyme probes from random-sequence DNA pools. We will also provide an in vitro selection protocol successfully used to derive RNA-cleaving fluorogenic DNAzyme probes that are capable of recognizing a targeted strain of Clostridium difficile.

Geniposide is a key iridoid glycoside from Gardenia jasminoides fructus widely used in traditional Chinese herbal medicine. However, detection of this small molecule represents a significant challenge mostly due to the lack of specific molecular recognition elements. In this study, we have performed in vitro selection experiments to isolate DNA aptamers that can specifically bind geniposide. Using a stringent selection procedure, we have isolated DNA aptamers that can distinguish geniposide from genipin and glucose, two structural analogs of geniposide. Two top aptamers exhibit low micromolar binding affinity towards geniposide, but show significantly reduced affinity to genipin and glucose. These aptamers have the potential to be further developed into analytical tools for the detection of geniposide.