A sensitive fluorescence biosensor based on ligation-transcription and CRISPR/Cas13a-assisted cascade amplification strategies to detect the H1N1 virus.

We propose a sensitive H1N1 virus fluorescence biosensor based on ligation-transcription and CRISPR/Cas13a-assisted cascade amplification strategies. Products are generated via the hybridization of single-stranded DNA (ssDNA) probes containing T7 promoter and crRNA templates to a target RNA sequence using SplintR ligase. This generates large crRNA quantities in the presence of T7 RNA polymerase. At such crRNA quantities, ternary Cas13a, crRNA, and activator complexes are successfully constructed and activate Cas13a to enhance fluorescence signal outputs. The biosensor sensitively and specifically monitored H1N1 viral RNA levels down to 3.23 pM and showed good linearity when H1N1 RNA concentrations were 100 pM-1 µM. Biosensor specificity was also excellent. Importantly, our biosensor may be used to detect other viral RNAs by altering the sequences of the two probe junctions, with potential applications for the clinical diagnosis of viruses and other biomedical studies.

Fluorescence biosensor to detect microRNAs via integrating DNA hairpins transition mediated strand displacement amplification with primer exchange reaction.

Herein, we constructed a fluorescence biosensor for the ultra-sensitive analysis of microRNAs (miRNAs) by combining DNA hairpins transition triggered strand displacement amplification (DHT-SDA) with primer exchange reaction (PER). Target miRNA initiated DHT-SDA to facilitate the generation of multiple single-stranded DNA (ssDNA) as PER primer, which was extended into a long ssDNA. The biosensor is successfully utilized in detecting miRNAs with high sensitivity (limit of detection for miRNA-21 was 58 fM) and a good linear relationship between 100 nM and 100 fM. By simply changing the DNA hairpin sequence, the constructed biosensor can be extended to analyze another miRNAs. Moreover, the biosensor has the feasibility of detecting miRNAs in real samples with satisfactory accuracy and reliability. Therefore, the fluorescent biosensor has great application potential in clinical diagnosis.

Ultrasensitive detection of microRNAs based on click chemistry-terminal deoxynucleotidyl transferase combined with CRISPR/Cas12a.

The specificity and sensitivity of microRNA (miRNA) detection play a vital role in the early diagnosis of cancer and the treatment of various diseases. Here, we constructed a fluorescent biosensor based on click chemistry-terminal deoxynucleotidyl transferase (ccTdT) combined with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)12a cascade amplification system to achieve ultrasensitive miRNA-21 detection. Target miRNA-21 was employed as a template for click chemistry ligation of two nucleic acid probes, the product of which can be combined with magnetic microbeads (MBs). Then the 3'-end of the ligated nucleic acid and complementary strand miRNA-21 was extended by TdT. The extended poly-T tails activated the trans-cleavage ability of CRISPR/Cas12a, cleaving the reporter gene to generate the fluorescent signal. The proposed biosensor has a wide linear detection range, from 1 pM to 105 pM, with detection limits as low as 88 fM under optimal experimental conditions. Hence, this fluorescent biosensor enables simple, sensitive detection of miRNAs and offers a promising analytical platform for clinical diagnostics and biomedical research.

Click Chemistry Actuated Exponential Amplification Reaction Assisted CRISPR-Cas12a for the Electrochemical Detection of MicroRNAs.

MicroRNAs (miRNAs) play a very important role in biological processes and are used as biomarkers for the detection of a variety of diseases, including neurodegenerative diseases, chronic cardiovascular diseases, and cancers. A sensitive point-of-care (POC) method is crucial for detecting miRNAs. Herein, CRISPR-Cas12a combined with the click chemistry actuated exponential amplification reaction was introduced into an electrochemical biosensor for detecting miRNA-21. The target miRNA-21 initiated the click chemistry-exponential amplification reaction in the electrochemical biosensor to produce numerous nucleic acid fragments, which could stimulate the trans-cleavage ability of CRISPR-Cas12a to cleave hairpin DNA electrochemical reporters immobilized on the electrode surface. Under optimal conditions, the minimum detection limit for this electrochemical biosensor was as low as 1 fM. Thus, the proposed electrochemical biosensor allows sensitive and efficient miRNA detection and could be a potential analysis tool for POC test and field molecular diagnostics.

CRISPR/Cas13a combined with hybridization chain reaction for visual detection of influenza A (H1N1) virus.

This study provides proof of concept of a colorimetric biosensor for influenza H1N1 virus assay based on the CRISPR/Cas13a system and hybridization chain reaction (HCR). Target RNA of influenza H1N1 virus activated the trans-cleavage activity of Cas13a, which cleaved the special RNA sequence (-UUU-) of the probe, further initiating HCR to copiously generate G-rich DNA. Abundant G-quadruplex/hemin was formed in the presence of hemin, thus catalyzing a colorimetric reaction. The colorimetric biosensor exhibited a linear relationship from 10 pM to 100 nM. The detection limit was 0.152 pM. The biosensor specificity was excellent. This new and sensitive detection method for influenza virus is a promising rapid influenza diagnostic test.

Ultrasensitive, rapid, and highly specific detection of microRNAs based on PER-CRISPR/CAS.

Abnormal microRNA (miRNA) expression levels are confirmed as diagnostic biomarkers of the emergence and development of diseases. In this study, we developed a fluorescence biosensor for detecting miRNAs based on double amplification reactions with the primer exchange reaction (PER) and CRISPR/Cas12a. In the absence of target miRNA-21, PER hairpins remained locked by the protector strands and the primers did not extend. In the presence of target miRNA-21, the miRNA-21 bound to the guard sequence and exposed primer binding sites. Also, the closed PER hairpin was unlocked to specifically extend primers into single-stranded DNA (ssDNA) of unequal lengths. These ssDNAs of unequal lengths could activate the cleavage of a reporter by Cas12a, leading to an increase in detectable fluorescence signals. A large number of short nucleic acid fragments were amplified by PER-CRISPR multiple cycle cleavage fluorescent probes. Based on PER-combined CRISPR/Cas12a established dual signal amplification method was characterized by a low limit of detection of 10fM. The fluorescent biosensor for miRNA detection had the advantages of low detection cost, simple operation, and mobility, providing a very promising platform for the point-of-care testing of miRNA-21.

Electrochemical biosensor for detecting pathogenic bacteria based on a hybridization chain reaction and CRISPR-Cas12a.

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.

An electrochemical biosensor for the detection of pathogenic bacteria based on dual signal amplification of Cu3(PO4)2-mediated click chemistry and DNAzymes.

A novel electrochemical biosensor for detecting pathogenic bacteria was designed based on specific magnetic separation and highly sensitive click chemistry. Instead of enzyme-antibody conjugates, organic-inorganic hybrid nanoflowers [concanavalin A (Con A)-Cu3(PO4)2] were used as the signal probe of the sandwich structure. The inorganic component, the copper ions of hybrid nanoflowers, was first used to amplify signal transduction for enzyme-free detection. Sodium ascorbate could dissolve Cu3(PO4)2 of the signal probe to produce Cu2+, which was subsequently converted to Cu+, triggering the Cu+-catalyzed alkyne-azide cycloaddition (CuAAC) reaction between azide-functionalized ssDNA (a fragment of the DNAzyme-containing sequence) and alkyne-functionalized ssDNA immobilized onto the electrode surface. As a result, the DNAzyme was immobilized onto the gold electrode, which produced a positive and stable electrical signal. An exceptional linear relationship was observed between the electrical signal and the concentration of Salmonella typhimurium (101-107 CFU mL-1) with a detection limit of 10 CFU mL-1. The developed electrochemical biosensor based on dual signal amplification of Cu3(PO4)2-mediated click chemistry and DNAzymes exhibited good results in detecting S. typhimurium in milk samples.

Visual assay of Escherichia coli O157:H7 based on an isothermal strand displacement and hybrid chain reaction amplification strategy.

Here, we describe a simple, sensitive, and enzyme-free method for visual point-of-care detection of 16S rRNA of Escherichia coli O157:H7 based on an isothermal strand displacement-hybrid chain reaction (ISD-HCR) and lateral flow strip (LFS). In this study, the secondary structure of 16S rRNA of E. coli O157:H7 was unwound by two helper oligonucleotides to expose the single-strand-specific nucleic acid sequence. The free specific sequence promoted the toehold-mediated strand displacement reaction to output a large number of FITC-labeled single-stranded DNA probes (capture probe [CP]). The 3'-end sequence of the reporter probe propagated a chain reaction of hybridization events between the two hairpin probes modified with biotin to form long nicked DNA polymers with multiple biotins (RP-HCR complexes); the free CP and RP-HCR complexes then form CP/RP-HCR complexes. The biotin-labeled double-stranded DNA CP/RP-HCR polymers then introduced numerous streptavidin (SA)-labeled gold nanoparticles (AuNPs) on the LFS. The accumulation of AuNPs produced a characteristic red band, which enabled visual detection of changes in the signal of 16S rRNA of E. coli O157:H7. The current approach could detect E. coli O157:H7 at concentrations as low as 102 CFU mL-1 without instrumentation. This approach thus provides a simple, sensitive, and low-cost tool for point-of-care detection of pathogenic bacteria, especially in resource-limited countries.

16S rRNA-functionalized multi-HCR concatemers in a signal amplification nanostructure for visual detection of Salmonella.

To prevent foodborne diseases and minimize their impacts, it is extremely important to develop a cost-effective and efficient bacterial detection assay for diagnostics, particularly in resource-poor settings. In this study, 16S rRNA from foodborne Salmonella was coupled with multiple HCR (hybridization chain reaction) concatemers and functionalized in a signal structure for lateral flow nucleic acid biosensor (LFNAB) detection. The 16S rRNA was incubated with two specific capture probes and multiple helper probes carrying the same initiator, to unwind its secondary structure and form an "initiators-on-a-string" complex. Through use of the initiators, each target 16S rRNA yielded multiple HCR concatemers tethered to numerous biotins, and numerous streptavidin-labeled gold nanoparticles were introduced on the LFNAB. The limit of detection was 53.65 CFU/mL for Salmonella. Notably, this method has high specificity and applicability for the detection of Salmonella in food and water samples.

A Colorimetric Immunosensor Based on Hemin@MI Nanozyme Composites, with Peroxidase-like Activity for Point-of-care Testing of Pathogenic E. coli O157:H7.

Recently, nanozymes have become a topic of particular interest due to their high activity level, stability and biocompatibility. In this study, a visual, sensitive and selective point-of-care immunosensor was established to test the pathogen Escherichia coli O157:H7 (E. coli O157:H7). Hemin and magainin I (MI) hybrid nanocomposites (Hemin@MI) with peroxidase-mimicking activities were synthesized via a "one-pot" method, involving the simple mixing of an antimicrobial peptide (MI) against E. coli O157:H7 and hemin in a copper sulfate sodium phosphate saline buffer. Hemin@MI nanocomposites integrating target recognition and signal amplification were developed as signal probes for the point-of-care colorimetric detection of pathogenic E. coli O157:H7. Hemin@MI nanocomposites exhibit excellent peroxidase activity for the chromogenic reaction of ABTS, which allows for the visual point-of-care testing of E. coli O157:H7 in the range of 102 to 108 CFU/mL, with a limit of detection of 85 CFU/mL. These data suggest this immunosensor provides accessible and portable assessments of pathogenic E. coli O157:H7 in real samples.

Immunoassay for foodborne pathogenic bacteria using magnetic composites Ab@Fe3O4, signal composites Ap@PtNp, and thermometer readings.

A point-of-care (POC) immunoassay was established for the sensitive and rapid detection of pathogenic Escherichia coli O157:H7, using magnetic Fe3O4 organic-inorganic composites (Ab@Fe3O4) for immunomagnetic separation, nanozyme platinum nanoparticle (PtNp) organic-inorganic composites (Ap@PtNp) for signal amplification, and thermometer readings. Antibodies and Fe3O4 were incubated in Cu2+ phosphate buffer to synthesize the magnetic composite Ab@Fe3O4 with antibodies, to specifically capture E. coli O157:H7. Antimicrobial peptides and PtNp were incubated in Cu2+ phosphate buffer to synthesize the signal composites Ap@PtNp with antimicrobial peptides (magainin I), recognizing and labeling E. coli O157:H7. In the presence of E. coli O157:H7, magnetic microcomposites targeted bacteria and signal microcomposites to form the sandwich structure: Ab@Fe3O4-bacteria-Ap@PtNp for magnetic separation. Ap@PtNp of signal composites catalyzed H2O2 to generate thermo-signals (temperature rise), which were determined by a thermometer. This point-of-care bioassay detected E. coli O157:H7 in the linear range of 101-107 CFU mL-1 and with a detection limit of 14 CFU mL-1. One-pot process magnetic Fe3O4 organic-inorganic composites (Ab@Fe3O4, magnetic microcomposites, MMC) for immunomagnetic separation and nanozyme platinum nanoparticle (PtNp) organic-inorganic composites (Ap@PtNp, signal microcomposites, SMC) were used as signal amplification and thermometer readings for E. coli O157:H7 detection.

Ferrocene-functionalized nanocomposites as signal amplification probes for electrochemical immunoassay of Salmonella typhimurium.

An electrochemical immunosensor based on ferrocene (Fc)-functionalized nanocomposites was fabricated as an efficient electroactive signal probe to amplify electrochemical signals for Salmonella typhimurium detection. The electrochemical signal amplification probe was constructed by encapsulating ferrocene into S. typhimurium-specific antimicrobial peptides Magainin I (MI)-Cu3(PO4)2 organic-inorganic nanocomposites (Fc@MI) through a one-step process. Magnetic beads (MBs) coupled with antibody were used as capture ingredient for target magnetic separation, and Fc@MI nanoparticles were used as signal labels in the immunoassays. The sandwich of MBs-target-Fc@MI assay was performed using a screen-printed carbon electrode as transducer surface. The immunosensor platform presents a low limit of detection (LOD) of 3 CFU·mL-1 and a linear range from 10 to 107 CFU·mL-1, with good specificity and precision, and was successfully applied for S. typhimurium detection in milk. Graphical abstract One-pot process antimicrobial peptides Magainin I-Cu3(PO4)2 organic-inorganic nanocomposites (Fc@MI) were used as ideal electrochemical signal label, integrating both essential functions of biological recognition and signal amplification. Screen-printed carbon electrode (SPCE) was used as the electrochemical system for Salmonella typhimurium detection.

An electrochemical aptasensor based on cocoon-like DNA nanostructure signal amplification for the detection of Escherichia coli O157:H7.

We developed an electrochemical aptasensor based on cocoon-like DNA nanostructures as signal tags for highly sensitive and selective detection of Escherichia coli O157:H7. The stable cocoon-like DNA nanostructures synthesized by the rolling circle amplification reaction were loaded with hemin as electrochemical signal tags to amplify the signals. The single-stranded DNA capture probes were modified on the surface of a Au electrode via a Au-S bond. The E. coli O157:H7 specific aptamer and capture probe formed double-stranded DNA structures on the Au electrode. The aptamer preferentially bound to E. coli O157:H7, causing the dissociation of some aptamer-capture probes and releasing some capture probes. Subsequently, the free capture probes hybridized with the DNA nanostructures through the cDNA sequence. Under optimal conditions, the change in the electrochemical signal was proportional to the logarithm of E. coli O157:H7 concentration, from 10 to 106 CFU mL-1, and the detection limit was estimated to be 10 CFU mL-1. The electrochemical aptasensor could be readily used to detect various pathogenic bacteria and to provide a new method of early diagnosis of pathogenic microorganisms.

A sensitive biosensor for determination of pathogenic bacteria using aldehyde dehydrogenase signaling system.

Aldehyde dehydrogenase (ALDH) was first developed as an enzymatic signaling system of a biosensor for sensitive point-of-care detection of pathogenic bacteria. ALDH and specific aptamers to Salmonella typhimurium (S. typhimurium), as organic components, were embedded in organic-inorganic nanocomposites as a biosensor signal label, integrating the functions of signal amplification and target recognition. The biosensing mechanism is based on the fact that ALDH can catalyze rapid oxidation of acetaldehyde into acetic acid, resulting in pH change with portable pH meter readout. The altered pH exhibited a linear relationship with the logarithm of S. typhimurium from 102 to 108 CFU/mL and detection limit of 46 CFU/mL. Thus, the proposed biosensor has potential application in the diagnosis of pathogenic bacteria.

An electrochemical biosensor based on methylene blue-loaded nanocomposites as signal-amplifying tags to detect pathogenic bacteria.

A sandwich-type electrochemical biosensor was successfully constructed for the sensitive detection of pathogenic bacteria. In this biosensor platform, methylene blue (MB) organic-inorganic nanocomposites (MB@MI) were synthesized from magainin I (MI, antimicrobial peptide specific to Escherichia coli O157:H7), Cu3(PO4)2 and MB via a one-pot method, and were explored as a novel electrochemical signal label of biosensors generating amplified electrochemical signals by differential pulse voltammetry (DPV). E. coli O157:H7 specifically sandwich bound to the aptamers on the electrode surface and MB@MI nanocomposites, and the changes in the current signal generated on the electrode surface were used for the quantitative determination of E. coli O157:H7. Under optimum conditions, the proposed biosensor showed excellent performance with a wide linear range of 102-107 CFU mL-1 and a low detection limit of 32 CFU mL-1, featuring favorable selectivity, repeatability and stability. According to the experiments conducted on real samples, the proposed approach is capable of detecting pathogenic bacteria in clinical diagnostics.

Sandwich immunoassay based on antimicrobial peptide-mediated nanocomposite pair for determination of Escherichia coli O157:H7 using personal glucose meter as readout.

A sandwich immunoassay was developed for determination of E. coli O157:H7. This is based on an antimicrobial peptide-mediated nanocomposite pair and uses a personal glucose meter as signal readout. The antimicrobial peptides, magainins I, and cecropin P1 were employed as recognition molecules for the nanocomposite pair, respectively. With a one-step process, copper phosphate nanocomposites embedded by magainins I and Fe3O4 were used as "capturing" probes for bacterial magnetic isolation, and calcium phosphate nanocomplexes composed of cecropin P1 and invertase were used as signal tags. After magnetic separation, the invertase of the signal tags hydrolyzed sucrose to glucose, thereby converting E. coli O157:H7 levels to glucose levels. This latter can be quantified by a personal glucose meter. Under optimal conditions, the concentration of E. coli O157:H7 can be determined in a linear range of 10 to 107 CFU·mL-1 with a detection limit of 10 CFU·mL-1. The method was successfully applied to the determination of E. coli O157:H7 in milk samples. Graphical abstract Schematic representation of sandwich immunoassay for E. coli O157:H7. One-pot synthetic of Fe3O4-magainins I nanocomposites (MMP) were used for magnetic capture. Cecropin P1-invertase nanocomposites (PIP) were used as signal tags. A personal glucose meter was used as readout to determine the target.

Point-of-care detection of 16S rRNA of Staphylococcus aureus based on multiple biotin-labeled DNA probes.

A visual method that combines multiple biotin-labeled DNA probes and lateral-flow nucleic acid biosensor was developed to detect Staphylococcus aureus. The 16S rRNA from Staphyloccocus aureus (S. aureus), coupled with multiple biotin-labeled DNA probes, was functionalized in a signal structure for lateral-flow point-of-care detection. The secondary structure of the 16S rRNA was unwound by two specific capture probes modified by Fam and multiple bridge probes, which extended additional sequences for use as initiators. By utilizing the initiators, each target 16S rRNA with multiple DNA probes could tether a number of biotin molecules, so that a large number of streptavidin-labeled gold nanoparticles could be introduced in the lateral flow assay. The images of the lateral flow detection results obtained using a smartphone were transmitted to a computer via Wi-Fi or Bluetooth connection for quantitative processing by ImageJ. The limit of detection was 103 cfu/mL without sample enrichment, and decreased to 0.12 cfu/mL following a 3-h enrichment of samples in growth medium. Notably, this method presented high specificity and applicability for the detection of S. aureus in food samples. In short, the developed visual non-specific operation method is very suitable for point-of-care diagnosis of pathogens in resource-limited countries.

Immunoassay for pathogenic bacteria using platinum nanoparticles and a hand-held hydrogen detector as transducer. Application to the detection of Escherichia coli O157:H7.

An innovative approach is presented for portable and sensitive detection of pathogenic bacteria. A novel synthetic hybrid nanocomposite encapsulating platinum nanoparticles, as a highly efficient catalyst, catalyzes the hydrolysis of the ammonia-borane complex to generate hydrogen gas. The nanocomposites are used as a label for immunoassays. A portable hand-held hydrogen detector combined with nanocomposite-induced signal conversion was applied for point-of-care testing of pathogenic bacteria. A hand-held hydrogen detector was used as the transducer. Escherichia coli O157:H7 (E. coli O157: H7), as detection target, formed a sandwich structure with magnetic beads and hybrid nanocomposites. Magnetic beads were used for separation of the sandwich structure, and hybrid nanocomposites as catalysts to catalyze the generation of hydrogen from ammonia-borane. The generated hydrogen was detected by a hydrogen detector using an electrochemical method. E. coli O157:H7 has a detection limit of 10 CFU·mL-1. The immunosensor made the hand-held hydrogen detector a point-of-care meter to be used outdoors for the detection and quantification of targets beyond hydrogen. Graphical abstract Schematic presentation of one-pot synthetic peptide-Cu3(PO4)2 hybrid nanocomposites embedded PtNPs (PPNs), encapsulating many Pt particles. The PPNs acts as an ideal immunoprobe for hand-held H2 detector signal readouts, by transforming pathogenic bacteria recognition events into H2 signals.

Disposable syringe-based visual immunotest for pathogenic bacteria based on the catalase mimicking activity of platinum nanoparticle-concanavalin A hybrid nanoflowers.

Disposable syringes were used in a novel point-of-care visual test for detecting pathogenic bacteria (Escherichia coli O157:H7 and Salmonella typhimurium). Hybrid nanoflowers composed of platinum nanoparticles and concanavalin A (Pt-nanoflowers) were prepared through a one-pot reaction and were found to be viable catalase mimics. They catalyze the decomposition of hydrogen peroxide (H2O2) to generate O2. When used as labels in immunoassays, they integrate both the functions of biological recognition and signal amplification. The disposable syringe pressure readout was combined with Pt-nanoflower signal conversion and successfully applied to a visual bacteria detection scheme. Both Escherichia coli O157:H7 and Salmonella typhimurium can be quantified with detection limits of as low as 15 and 7 CFU·mL-1, respectively. Graphical abstract One-pot synthetic platinum nanoparticle (PtNP)-concanavalin A hybrid nanoflowers (Pt-nanoflowers), have been used as ideal signal labels for immunoassays and integrating both essential functions of biological recognition and signal amplification. Disposable syringes were used as a readout to detect pathogenic bacteria.