Search

DNA walkers, as intelligent artificial DNA nanomachines, have been widely used as efficient nucleic acid amplification tools that the detection sensitivity can be improved by incorporating DNA walkers into DNA biosensors. Nevertheless, since the premature release or flameout in a region of locally exhausted substrate, the walking efficiency of DNA walkers remains unsatisfactory. In this work, we design a smart tripedal DNA walker that is formed by target-initiated catalyzed hairpin assembly (CHA), which can move along the DNA duplex tracks on electrode driven by toehold-mediated DNA strand displacement (TMSD) for transduction and amplification of electrochemical signals. Emphatically, this flexible tripedal DNA walker is capable of walking freely along the tracks with unconstrained walking range. Moreover, the design of multi-legged walker can weaken the derailment of leg DNA and shorten the moving time on electrode, ensuring the processive walking with high efficiency. Additionally, the persistent walking of tripedal walker is driven by cascading TMSD, which eliminates the defects of high cost and instability of enzyme-assisted amplification technology. Therefore, the tripedal DNA walker-based electrochemical biosensor has enormous potential for the applications of OTA detection, and reveals a new avenue for food safety analysis and clinical diagnosis.

A novel fluorescence sensing strategy for ultrasensitive and highly specific detection of adenosine triphosphate (ATP) has been developed by the combination of the proximity ligation assay with bidirectional enzymatic repairing amplification (BERA). The strategy relies on proximity binding-triggered the release of palindromic tail that initiates bidirectional cyclic enzymatic repairing amplification reaction with the aid of polymerase and two DNA repairing enzymes, uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV). A fluorescence-quenched hairpin probe with a palindromic tail at the 3' end is skillfully designed that functions as not only the recognition element, primer, and polymerization template for BERA but also the indicator for fluorescence signal output. On the basis of the amplification strategy, this biosensor displays excellent sensitivity and selectivity for ATP detection with an outstanding detection limit of 0.81 pM. Through simultaneously enhancing the target response signal value and reducing nonspecific background, this work deducted the background effect, and showed high sensitivity and reproducibility. Moreover, our biosensor also shows promising potential in real sample analysis. Therefore, the proximity-enabled BERA strategy indeed creates a simple and valuable fluorescence sensing platform for ATP identification and related disease diagnosis and biomedical research.

We have developed a novel amplification strategy termed Endo IV-assisted feedback amplification (EFA) taking advantages of rolling circular amplification (RCA) and Endo IV-assisted signal amplification (ESA) biosensing platform for detecting of adenosine triphosphate (ATP). Two kinds of specially programmed DNA complexes were employed into EFA system, one composed of a split aptamer fragment and a circular template, and the other composed of AP probe and the same circular template. Hence, ATP as a target induced the self-assembly of spilt aptamer fragments and initiated RCA reaction generating a linear DNA, which consists of hybridization elements with Complex II and formation elements of G-quadruplex. More importantly, the addition of endonuclease IV can cut the Complex II into two parts, and one of which can be trimming by phi29 DNA polymerase initiating the new round of RCA reaction producing more RCA products. Thus significantly enhanced fluorescent signal can be measured for ATP as expected, and our proposed strategy exhibits improved performances toward ATP ultrasensitive detection with a limit of detection (LOD) as low as 0.09 nM. Additionally, our developed biosensor demonstrates high selectivity and the superiority of simplicity towards ATP. Above these significant aspects, our proximity binding-induced RCA reaction-based fluorescent assay and Endo IV-fueled feedback signal amplification strategy presents an optimal detection performance towards ATP for potential application in related research and clinical diagnosis.

Herein, a split G-quadruplex DNAzyme as a signal reporter was integrated into an electrochemical sensing platform for the detection of antibiotics with specificity and sensitivity. To improve the signal-to-noise ratio, two G-rich oligonucleotide sequences (G1 and G2) were blocked into two different hairpin probes, preventing the two segments from assembling into a spilt G-quadruplex structure. Moreover, we designed a double-arch probe, consisting of an aptamer as the recognition element and two-step enzymatic signal amplification. Concretely, the first is the Nt.BbvCI-assisted nicking cyclic reaction activated by target-aptamer binding, and the second is exonuclease III-aided cyclic amplification for generating abundant G1 and G2. The modified capture probe on the electrode was used to combine G1 and G2 to form the spilt G-quadruplex/hemin when K+ and hemin were present. This complex plays the role of DNAzyme with superior horseradish peroxidase activity in catalyzing the decomposition of H2O2. Under optimal conditions, this biosensor showed an excellent performance for sensing kanamycin with a detection limit of 83 fM for kanamycin concentrations ranging from 100 fM to 1 nM. Hence, the proposed strategy has potential as an efficient and actual platform for small molecule analysis.

A facile signal-on electrochemical DNA biosensor has been developed for ultrasensitive detection of pathogenic bacteria using an Exo III-assisted autonomous multiple-cycle amplification strategy. The strategy relies on pathogens and aptamer binding-initiated release of a trigger, which combines with the 3′-protruding terminus of the hairpin probe 1, leading to the formation of double-stranded DNA with a blunt 3′ terminus which starts the Exo III-assisted multiple signal amplification reaction. In addition, hairpin probe 2 labeled with an electroactive reporter at the middle of the loop region is ingeniously designed to contain a short hairpin-embedded segment, which can fold into a hairpin structure via an Exo III-assisted cleavage reaction, thus bringing the redox molecule in proximity to the electrode surface for “signal-on” sensing. Under optimal conditions, this biosensor exhibits a very low detection limit as low as 8 cfu mL−1 and a wide linear range from 1.0 × 101 to 1.0 × 107 cfu mL−1 of target pathogenic bacteria. As far as we know, this is the first time that the Exo III-assisted autonomous multiple-cycle amplification strategy has been used for signal-on electrochemical sensing of pathogenic bacteria. In addition, the proposed sensor can also be used for highly sensitive detection of other targets by changing the aptamer sequence, such as nucleic acids, proteins and small molecules. Therefore, the proposed signal-on electrochemical sensing strategy might provide a simple and practical new platform for detection of pathogenic bacteria and related biological analysis, food safety inspection and environmental monitoring.

We have formulated a rapid and high-efficiency fluorescent biosensing platform based on a target-activated triple-helix molecular switch (THMS)-conformation-change-induced exponential rolling circular amplification (RCA) strategy for the ultrasensitive detection of miR-21. In this strategy, there are several aspects that are worthwhile. First, the functionalized THMS, comprising a typical triplex structure (T-A·T), specific recognition sequence for nicking endonuclease, complementary sequence for miR-21, and RCA product-annealing sequence, was concurrently used to perform signal transduction with one fluorophore and one quencher. As compared to the traditional double-helix molecular switches or molecular beacons, one of the biggest differentiating factors is that the properties of THMSs are independent of any specific binding sequence that they may contain. As far as we know, this is the first time that an ingeniously designed THMS not only contains the primer for exponential RCA, but also functions as the tracer for fluorescence assay. In the presence of miR-21, targets can induce conformation changes in THMS with the release of the trapped DNA segment (P), which, in turn, can activate the first run of the RCA process. Meanwhile, the RCA reaction is also initiated by the formation of a similar primer (SP) as the trapped DNA through a continuous "extension-nicking" reaction. Secondly, the resultant first-generation RCA product consists of numerous tandem repeated regions that can attach to countless THMSs, resulting in the release of the trapped DNA segment (P) for initiating the second run of the RCA reaction. Significantly, a large amount of THMSs were continuously consumed to yield a remarkably strong fluorescent signal. In addition, this biosensor was demonstrated to exhibit improved sensitivity owing to the high efficiency and rapid amplification kinetics of the exponential RCA and high selectivity toward miR-21 with a limit of detection as low as 1.1 aM. Hence, the target-mediated THMS-conformation-change-initiated exponential RCA strategy presents an optimal detection performance toward analytes for potential applications in related fundamental research and clinical diagnosis.

A simple and robust fluorescence sensing strategy has been developed for the detection of pathogenic bacteria by the combination of the dual functionality of phi29 DNA polymerase with isothermal circular strand displacement polymerization (ICSDP). The strategy relies on target-triggered formation of a mature primer that initiates the cyclic strand displacement polymerization reaction with the aid of dual functional phi29; thus, amplified detection of the target can be achieved. To our knowledge, this work is the first report where dual functional phi29-assisted ICSDP has been employed for fluorescence sensing of pathogenic bacteria. It is worth noting that a hairpin pre-primer is introduced that can be trimmed into a mature primer for initiating ICSDP via the 3' → 5' proofreading exonuclease activity of phi29, which contributes to the ultrahigh specificity of the strategy owing to the elimination of the unwished nonspecific extension. On the basis of the present amplification strategy, our biosensor exhibits excellent specificity and sensitivity toward S. typhimurium with an excellent detection limit as low as 1.5 cfu mL-1. In addition, the strategy offers the advantages of a simplified operation, shortened analysis time, and highly sensitive detection of pathogens with only a one-step reaction. Furthermore, by redesigning the corresponding binding molecules, the proposed strategy can be easily extended for the detection of a wide spectrum of analytes. Hence, the dual functional phi29-assisted ICSDP strategy indeed creates a robust and convenient fluorescence sensing platform for the identification of pathogenic bacteria and related food safety analysis.

DNA walkers, one of the artificial molecular machines which are constructed via smart synthetic DNA, have attracted rapidly growing attention from researchers in the biosensing field. In this work, we design an Exonuclease III (Exo III)-aided target-aptamer binding recycling (ETBR) activated bipedal DNA machine for highly sensitive electrochemical detection of antibiotics. To the best of our knowledge, this is the first time that a bipedal DNA machine has been applied in electrochemical sensing for antibiotics. On the one hand, the bipedal DNA walker exceeds the conventional single swing arm DNA walker in terms of walking efficiency and stability. On the other hand, the ETBR strategy, along with efficient strand displacement amplification via stepwise movement of a bipedal DNA walker significantly promotes the signal amplification efficiency. Under optimal conditions, this bipedal DNA machine possesses a detection limit of 7.1 fM within a linear detection range from 10 fM to 100 pM. Moreover, this electrochemical biosensor is expected to detect a wide variety of analytes using the corresponding target recognition probes. Thus, our proposed strategy offers a highly efficient, stable and practical platform for small molecule analysis.

A novel fluorescence biosensing strategy for ultrasensitive and specific detection of pathogenic bacteria based on target-triggered enzymatic repairing amplification (ERA) has been developed. This strategy relies on target–aptamer binding mediated ERA reaction, which is carried out cyclically with the help of polymerase and two DNA repairing enzymes, uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV) to produce amplified fluorescence signal. In our assay, the specially designed hairpin probe (HAP) is used as DNA template responsible for producing a great quantity of reporter oligonucleotides and secondary primers, which can initiate a new cycle of polymerization-repairing amplification. Moreover, by the combination of polymerase-catalyzed incorporation of lesion bases with UDG and Endo IV-assisted ERA, multiple cycle of amplification of the recognition event is achieved, enabling ultrasensitive detection of pathogenic bacteria. Under optimal conditions, this biosensor exhibits ultrasensitivity toward target pathogenic bacteria with detection limits of 9.86 cfu mL−1 and a detection range of 5 orders of magnitude. Additionally, the biosensor has the ability of combating nonspecific background. Furthermore, an archer probe containing the anti-target aptamer sequence and a primer sequence is designed, which translates the binding of target to aptamer into the presence of primer sequence, enabling the detection of various targets, such as protein, DNA, small molecular, and any substance possessing its aptamer. Hence, the proposed target-triggered ERA-based signal-on fluorescence sensing strategy indeed create a versatile and useful platform for detection of pathogenic bacteria, related food safety analysis and clinical diagnosis.

The authors describe a label-free electrochemical aptasensor for ultrasensitive and highly specific detection of the antibiotic based on cascade enzymatic recycling couple with DNAzyme amplification. The assay involves two sequential reactions: the first reaction is a λ exonuclease-assisted cyclic digestion reaction triggered by target-aptamer binding. The second reaction is a nicking endonuclease-aided cyclic nicking reaction, which produces a large amount of G-rich nucleic acid segments. These form a G-quadruplex/hemin complex in the presence of K(I) ions and hemin. Because the G-quadruplex/hemin complex acts as a horseradish peroxidase-mimicking DNAzyme with excellent redox activity, the electrochemical signal transduction is accomplished due to the electroreduction of H2O2. It appears that this work is the first example that cascade enzymatic recycling coupled to DNAzyme amplification is used for antibiotic detection. The aptasensor was applied to the quantitation of kanamycin and gave a response that is linear in the 1 pM to 10 nM kanamycin concentration range, with a detection limit as low as 0.5 pM. The working voltage (vs. Ag/AgCl) at which data can be acquired best is −0.35 V. The assay offers the advantages of remarkably improved sensitivity, use of affordable instrumentation, and simplified operation without the need for electrochemical labeling or addition of labile reagents. Thus, cascade enzymatic recycling coupled to DNAzyme amplification represents a versatile platform for highly sensitive and specific antibiotics detection.

The authors describe a colorimetric method for the determination of Hg(II) ion. It is based on the color change from red to colorless as displayed by gold nanoparticle (AuNP) modified with thymine - rich DNA. Signal amplification is accomplished by free strand displacement recycling. In this strategy, Hg(II) unfolds the arch-trigger duplex due to the high affinity between Hg(II) and the thymines to form T-Hg(II)-T structures, thereby causing the release of trigger. The liberated trigger unfolds the hairpin structure of H1, and unfolded H1 further unfolds with H2. As a result, the H2 hairpin displaces trigger, and the released trigger unfolds another H1. This results in strong and enzyme-free strand displacement recycling amplification. The aggregation of DNA-AuNPs occurs in the presence of the duplex formed by hairpins H2 and H1. This results in a color change from red to colorless that can be visually observed. Under optimal conditions, the assay has a detection range over 4 orders of magnitude and a 3.4 nM detection limit. The assay is selective, sensitive, rapid and cost-effective. In our perception, it represents a useful platform for determination of Hg(II).

In this study, a novel electrochemical biosensor based on exonuclease III (Exo III)-aided autonomous cascade signal amplification for ultrasensitive and highly specific detection of Salmonella typhimurium (S. typhimurium) has been reported. Herein, two duplex DNA probes were ingeniously designed. An Ap–Pr duplex probe was constructed as a recognition element by hybridizing an S. typhimurium aptamer (Ap) with a primer (Pr). A P–S duplex probe has been designed to contain a protruding DNA fragment at 5′-termini that has an identical sequence with Pr (the secondary Pr), a complementary duplex DNA that is used for blocking a signal-triggered probe (S), and an overhung nucleic acid segment at 3′-end that can anneal with Pr and form double-stranded DNA with a blunt 3′-terminus, thus initiating Exo III-aided amplification reaction. Upon sensing of the target S. typhimurium, the specific recognition of S. typhimurium and aptamer releases Pr, which anneals with the P–S duplex and triggers the Exo III cleavage reaction, accompanied by release of Pr, S, and secondary Pr. After this, the released Pr and secondary Pr anneal with other P–S duplex, which in turn triggers the Exo III-assisted cycling cleavage. Thus, large amounts of S are liberated, which hybrids with S* (DNA probe complementary to S); thereby, 5′-methylene blue (MB)-labelled DNA probe (MBP) folds into a hairpin structure with 5′-terminus close to electrode surface for efficient electronic transfer. Moreover, the S–S* hybrid duplex is specifically hydrolyzed by Exo III to liberate S, which accordingly results in the cycling cleavage of S*. Therefore, numerous MBP transform into a hairpin structure with electroactive MB close to the electrode surface; this generates significantly amplified electrochemical signal towards S. typhimurium detection. The present developed strategy exhibited high sensitivity towards S. typhimurium with a detection limit of 2.8 × 10 cfu mL−1. Furthermore, the proposed autonomous cascade signal amplification strategy can be easily extended for detection towards a wide spectrum of analytes including nucleic acid, proteins, and small molecules possessing their corresponding aptamers. Therefore, the developed Exo III-aided autonomous cascade signal amplification strategy might indeed provide a facile and universal tool for bioanalysis and clinical diagnosis.

We report the development of a novel fluorescence biosensing strategy based on branched-rolling circle amplification (RCA) coupled with nicking endonuclease signal amplification (NESA) for the ultrasensitive and highly specific detection of miRNA. This biosensor relies on target miRNA-triggered quadratic RCA and endonuclease-assisted cyclic nicking of a molecular beacon (MB). The specific binding between target miRNA and hairpin probe 1 (HAP1) leads to a change in the conformation of HAP1, releasing an exposed DNA segment that can hybridize with circular probe 1 and subsequently induce a primary RCA reaction. The generated RCA product combines specifically to hairpin probe 2 (HAP2), resulting in unfolded HAP2 and liberating a new exposed DNA segment that can initiate quadratic RCA. Then MB hybridizes with the tandem repeated DNA segments produced by quadratic RCA, forming the specific nicking site of the nicking endonuclease. Therefore, the nicking endonuclease cleaves MB into two pieces, liberating a strong fluorescence signal. Meanwhile, the quadratic RCA products initiate the next cycle of hybridization and cleavage, thereby acquiring a significantly increased fluorescence signal. Using the branched-RCA coupled with the NESA strategy, an ultrasensitive miRNA-155 assay method has been developed with a wide linear range from 5 aM to 500 fM and a low detection limit of 3.9 aM. This work is, to our knowledge, the first report that branched-RCA coupled with NESA has been used for the fluorescence assay of miRNA. The developed biosensing strategy might hold great potential for detecting miRNA and for early diagnosis in miRNA-related diseases.

An amplified electrochemical biosensing scheme is described for lead(II) ions. It is making use of DNAzyme-assisted target recycling and catalytic hairpin assembly (CHA). The hairpin strand (substrate probe for the Pb2+-based DNAzyme; referred to as SP) is composed of trigger probe (TP) and a capture probe 1 attached to gold nanoparticles (AuNP). In the presence of the enzyme probe that partially hybridizes with SP, the introduction of Pb2+ triggers target recycling and drives the highly amplified translation of target Pb(II) to TP. The CHA reaction is further initiated by TP. The modified AuNP act as the connecting unit, and this leads to the formation of a 3D DNA-AuNP network on the electrode (which is the third amplification step). It can bind the positively charged redox mediator RuHex via electrostatic interaction for electrochemical detection. This biosensor has a low detection limit (95 pM) and any analytical range that covers the 100 pM to 5 μM Pb(II) concentration range. It is selective over other divalent metal ions. It was applied to the determination of Pb2+ in spiked real-world samples.

A rapid and label-free fluorescence biosensing strategy for highly sensitive detection of microRNA-122 (miR-122) has been developed by the combination of DNA three-way junction (TWJ)-actuated strand displacement and a fluorescence light-up Ag nanocluster (AgNC) probe. In the presence of target miR-122, the attachment of miR-122 to its complementary DNA results in the unblocking of the toehold and branch migration domains in the TWJ, activating the strand displacement reaction (SDR) accompanied by the proximity between the G-rich DNA probe and DNA-AgNC probe; thus a remarkably enhanced fluorescence signal of AgNCs can be obtained owing to the G-rich fluorescence enhancement mechanism. The results reveal that this biosensor exhibits superb specificity and high sensitivity toward miR-122 with a detection limit of 0.030 nM. In addition, the practicality of the biosensor is demonstrated by analyzing miR-122 in three cell lines with satisfactory results. Furthermore, by the utilization of the toehold-mediated SDR and DNA-AgNC conjugates, this proposed strategy offers the advantages of rapidness, convenience, low cost, and simplified operation without the need for biological labeling and the addition of enzymes. Thus, the constructed biosensor might provide a valuable and practical tool for detecting miRNA and the related clinical diagnosis and fundamental biomedicine research.

DNA can be configured into unique high-order structures due to its significantly high programmability, such as a three-way junction-based structure (denoted Y-shaped DNA), for further applications. Herein, we report a label-free fluorescent signal-on biosensor based on the target-driven primer remodeling rolling circle amplification (RCA)-activated multisite-catalytic hairpin assembly (CHA) enabling the concurrent formation of Y-shaped DNA nanotorches (Y-DNTs) for ultrasensitive detection of ochratoxin A (OTA). Two kinds of masterfully-designed probes, termed Complex I and II, were pre-prepared by the combination of a circular template (CT) with an OTA aptamer (S1), a substrate probe (S2) and hairpin probe 1 (HP1), respectively. Target OTA specifically binds to Complex I, resulting in the release of the remnant element in S2 and successive remodeling into a mature primer for RCA by phi29 DNA polymerase, thus a usable primer-CT complex is produced, which actuates primary RCA. Then, numerous Complex II probes can anneal with the first-generation RCA product (RP) with multiple sites to activate the CHA process. With the participation of endonuclease IV (Endo IV) and phi29, HP1 as a pre-primer containing a tetrahydrofuran abasic site mimic (AP site) in Complex II is converted into a mature primer to initiate additional rounds of RCA. So, countless Y-DNTs are formed concurrently containing a G-quadruplex structure that enables the N-methylmesoporphyrin IX (NMM) to be embedded, generating remarkably strong fluorescence signals. The biosensor was demonstrated to enable rapid and accurate highly efficient and selective detection of OTA with an improved detection limit of as low as 0.0002 ng mL-1 and a widened dynamic range of over 4 orders of magnitude. Meanwhile, this method was proven to be capable of being used to analyze actual samples. Therefore, this proposed strategy may be established as a useful and practical platform for the ultrasensitive detection of mycotoxins in food safety testing.

A colorimetric biosensor and visual test is described for the determination of mercury(II). It relies on the specific thymine-Hg(II)-thymine (T-Hg2+-T) interaction which induces a cyclic amplification process (caused by the enzyme exonuclease III) and the aggregation of gold nanoparticles. These results in a color change from red to violet. Under optimized conditions, this colorimetric assay (best performed at 524 nm) has a detection limit as low as 0.9 nM with a detection range over 4 orders of magnitude (from 1 nM to 10 μM).

In the work, a signal-on electrochemical DNA sensor based on multiple amplification for ultrasensitive detection of antibiotics has been reported. In the presence of target, the ingeniously designed hairpin probe (HP1) is opened and the polymerase-assisted target recycling amplification is triggered, resulting in autonomous generation of secondary target. It is worth noting that the produced secondary target could not only hybridize with other HP1, but also displace the Helper from the electrode. Consequently, methylene blue labeled HP2 forms a “close” probe structure, and the increase of signal is monitored. The increasing current provides an ultrasensitive electrochemical detection for antibiotics down to 1.3 fM. To our best knowledge, such work is the first report about multiple recycling amplification combing with signal-on sensing strategy, which has been utilized for quantitative determination of antibiotics. It would be further used as a general strategy associated with more analytical techniques toward the detection of a wide spectrum of analytes. Thus, it holds great potential for the development of ultrasensitive biosensing platform for the applications in bioanalysis, disease diagnostics, and clinical biomedicine.

A novel label-free, low cost electrochemical biosensor for highly sensitive and selective detection of E. coli has been developed based on rolling circle amplification (RCA) coupled silver nanoclusters (AgNCs) as an effective electrochemical probe. This strategy is to our knowledge the first approach where the combination of RCA with the efficient catalytic property of AgNCs has been applied in bioanalysis. Our biosensor relies on sandwich type immuno-recognition and an in situ RCA reaction, which results in the enrichment of functional oligonucleotide probe (FOP) encapsulated AgNCs. Due to the high electrocatalytic activity of AgNCs toward H2O2 reduction, stable and sensitive electrochemical signals can be achieved from the biological recognition events. The results reveal the calibration plot obtained for E. coli O157:H7 is approximately linear from 3.7 × 10 to 3.7 × 106 cfu mL−1 with a limit of detection of 31 cfu mL−1. This label-free biosensor has advantages over conventional electrochemical assays in its significantly improved sensitivity, remarkably widened dynamic range, and substantially lowered background signal. Moreover, the proposed biosensor has been demonstrated to allow accurate quantitative assay of E. coli O157:H7 in milk samples. By virtue of these merits, our RCA coupled AgNCs-based electrochemical biosensor might create a useful and valuable tool for the detection of E. coli O157:H7 and related molecular diagnostics in food safety analysis.

In this work, a simple, isothermal, and ultrasensitive homogeneous colorimetric sensor for pathogenic bacteria detection has been developed on the basis of target-triggered exponential amplification reaction (EXPAR). An aptamer–primer probe (arched probe) containing anti-target aptamer and a primer sequence, which is released under the challenging of target, is used for recognizing target and triggering EXPAR-based polymerase elongation. Due to EXPAR coupled DNAzyme amplification strategy, the presence of target pathogenic bacteria leads to the formation of numerous G-quadruplex oligomers in solution, which folds into G-quadruplex/hemin complexes with the help of K+ and hemin, thus generating extremely strong catalytic activity toward H2O2 and giving a remarkably strong UV-vis absorption. This work is a novel design that EXPAR coupled DNAzyme amplification technique has been integrated into colorimetric assay for detecting pathogenic bacteria. Under optimal conditions, the proposed biosensor exhibits ultrahigh sensitivity toward target pathogenic bacteria with detection limits of 80 cfu mL−1. Besides, our biosensor also shows high selectivity toward target pathogenic bacteria and has the advantages in its low cost, simplified operations without the need of labeling steps and additional labile reagents. Hence, the EXPAR coupled DNAzyme amplification-based colorimetric method might create a useful and practical platform for detecting pathogenic bacteria and related food safety analysis and environmental monitoring.

In this work, a novel electrochemical DNA sensor based on exonuclease III (Exo III)-assisted autocatalytic DNA biosensing platform for ultrasensitive detection of antibiotics has been reported. When the arched probe was challenged with target, the released primer caused by the specific recognition of the aptamer and target antibiotics hybridized with the 3′-protruding terminus in the HP1. Then, Exo III could catalyze the stepwise removal of mononucleotides from this flat terminus, resulting in the release of the primer and trigger. As a secondary target, trigger could replace Helper from the 5′-MB labeled HP2, and HP2 formed a “close” probe structure, confining MB close to the electrode surface for efficient electron transfer. To our best knowledge, such work is the first report about Exo III-assisted autocatalytic DNA biosensing platform combing with signal-on sensing strategy, which has been utilized for quantitative determination of antibiotics. It would be further used as a general strategy associated with more analytical techniques toward the detection of a wide spectrum of analytes. Thus, it holds great potential for the development of ultrasensitive biosensing platform for the applications in bioanalysis, disease diagnostics, and clinical biomedicine.

A trimetallic dendritic Au@PdPt nanparticles (Au@PdPt NPs) with intrinsic peroxidase-like catalytic activity were fabricated by a facile method. Au@PdPt NPs catalyzed the reduction of H 2 O 2 with the help of the electron mediator (thionine), which produced the electrochemical signals dramatically amplified. A sandwiched sensor was designed and implemented in the paper chip device for the determination of K-562 cells. The surface of dendritic Au@PtPd NPs was linked with folic acid by click chemistry. Folic acid was attached to the folate receptor (FR) of cell surface with high affinity. So, K-562 cells could be assayed based on folic acid-functionalized Au@PtPd NPs. On the other hand, folic acid-functionalized Au-paper working electrode on origami electrochemical device was fabricated, which could be used to capture K-562 cells. The twice utilization of folic acid for the capture of K-562 cells as well as the attachment of dendritic Au@PtPd NPs nanoprobe, which enhanced the selectivity of sensor for the determination of cells. The proposed biosensor exhibited excellent analytical performance toward the cytosensing of K562 cells with a wide detection linear range from 1.0 × 10 2 to 2.0 × 10 7 cells mL −1 and a detection limit of 31 cells mL −1 . Further, the proposed electrochemical sensor was applied to monitor cells. Au@PtPd NPs nonenzymatic catalysts could be used as signal-amplifying nanoprobes for ultrasensitive electrochemical sensing.

The authors describe a fluorometric strategy for the detection of pathogenic bacteria with ultrasensitivity and high specificity. This strategy relies on the combination of target-modulated photoinduced electron transfer (PET) between G-quadruplex DNAzyme and DNA (labeled with silver nanoclusters) along with hairpin probe-based circular exponential amplification. The reaction system involves three hairpin probes (H1, H2 and H3). Probe H1 contains an aptamer against S. Typhimurium and the recognition sequence for nicking endonuclease. It is used to recognize S. Typhimurium and participates in polymerase-catalyzed target recycle amplification and secondary-target recycle amplification. Probe H2 contains an aptamer against hemin and is used to form the G-quadruplex DNAzyme in the presence of hemin and potassium ion. It acts as the electron acceptor and quenches the fluorescence of the labeled DNA. Fluorescence is best measured at excitation/emission wavelengths of 567/650 nm. Probe H3 contains the template sequence for the synthesis of AgNCs and the H2-annealing sequence. Both H2 and H3 are utilized to perform a strand displacement reaction and to achieve PET between G-quadruplex DNAzyme and DNA/AgNCs. To the best of our knowledge, this is the first example of a PET between G-quadruplex DNAzyme and DNA/AgNCs coupled with circular exponential amplification. The assay has an ultra-low detection limit 8 cfu·mL−1 of S. Typhimurium. The assay is rapid, accurate, inexpensive and simple. Hence, the strategy may represent a useful platform for ultrasensitive and highly specific detection of pathogenic bacteria as encountered in food analysis and clinical diagnosis.

Herein, we have reported the development of a simple, rapid, and low cost colorimetric method for the detection of antibiotic based on target-activated split peroxidase DNAzyme coupled with dual nicking enzyme signal amplification (NESA). To lower background signal in G-quadruplex DNAzyme-based detection, the two split G-rich parts are caged into two different hairpin probes, respectively, preventing the two parts from assembling into the G-quadruplex structure. By the combination of restriction endonuclease-assisted cleavage reaction with the spilt G-quadruplex probes, target-modulated release of the two split G-rich parts is achieved, affording high specificity of antibiotic detection. Our strategy features with several aspects. First, the less background signal produced by the self-assembly of G-quadruplex in the absence of target is effectively eliminated owing to the pre-blocking of the two split G-rich parts. Second, dual NESA coupled G-quadruplex DNAzyme amplification strategy is integrated with colorimetric assay of antibiotic, which significantly improves the detection sensitivity. Third, peroxidase-mimicking DNAzyme is used as biocatalyst in our reaction system, which can catalyze the oxidation of 2,2' - azino - bis (3 - ethylbenzothiozoline - 6 - sulfonic acid) (ABTS2-) mediated by H2O2 to generate the colored radical anion (ABTS•-), allowing to low cost and visual detection of antibiotic by the naked eye. Under optimized conditions, the results revealed the proposed biosensor exhibits excellent specificity and sensitivity toward kanamycin with a detection limit as low as 14.7 pM. Hence, the target-activated split G-quadruplex DNAzyme and dual NESA-based strategy provides a useful and practical platform for antibiotic residues determination and other analytes detection in bio-analysis.

An RNA aptamer-based electrochemical biosensing strategy has been developed for sensitive and selective detection of malachite green (MG). This biosensor is fabricated by the self-assembly of a thiolated MG aptamer (MGA) on AuNPs/graphene–chitosan nanocomposite modified glass carbon electrode. In addition, a short alkanethiol is further assembled on the AuNPs surface to generate uniform packing and reduce nonspecific adsorption. When the modified electrode is incubated in the presence of MG, MGA combines specifically with MG, which causes the horseradish peroxidase (HRP)-labelled MG antibody close to the electrode surfaces. As a result, MG detection is realized by outputting a redox current from electro-reduction of the hydrogen peroxide reaction catalyzed by HRP. Differential pulse voltammetry (DPV) is performed to record the signal responses. The results reveal the biosensor displays a very low detection limit as low as 16.3 pg mL−1 and a wide linear range from 1 × 10−4 to 10 μg mL−1 of MG. Hence, this proposed RNA aptamer-based electrochemical strategy may offer a simple, rapid, cost-effective, highly selective and sensitive method for the quantification of MG.

Herein, an Exonuclease III (Exo III)-powered DNA walking machine has been integrated into electrochemical sensing for label-free and highly sensitive detection of antibiotics. The electrochemical sensing interface is prepared by co-assembly of hemin aptamer-containing DNA track (DT) and DNA walker (DW)-locking probe (LP) duplex on gold electrode surface. The specific target and aptamer interaction triggers Exo III-assisted cyclic amplification, releasing the unlocking probe (UP) that is used for the deblocking of DW. Then the autonomous moving of DW on electrode surface is propelled via Exo III digestion of hybridized DT. The consumption of DT induces the formation of G-quadruplex/hemin DNAzyme that can catalyze the reduction of H2O2, thus a significantly strong current signal is released. Using ampicillin as a model analyte, the proposed biosensor achieves a detection limit of 0.76 pM within a linear detection range from 1 pM to 10 nM. Additionally, this biosensing strategy can be readily extended for the detection of wide variety of analytes by using corresponding target recognition probes. Therefore, the proposed strategy indeed creates a highly sensitive and label-free electrochemical sensing platform for molecular diagnosis applications.

Platinum nanoparticles (Ptnano) were used in combination with multi-walled carbon nanotubes (MWCNTs) for fabricating sensitivity-enhanced electrochemical l -lactate biosensor. The composite film of MWCNTs and Ptnano was dispersed on the surface of the glassy carbon electrode (GCE). l -lactate oxidase (LOD) was immobilized on MWCNTs/Ptnano/GCE surface by adsorption. The resulting LOD/MWCNTs/Ptnano electrode was covered by a thin layer of sol–gel to avoid the loss of LOD in determination and to improve the anti-interferent ability. Moreover, the sol–gel microenviroment contributes to both intensified stability and permselectivity. The cyclic voltammetry results indicated that MWCNTs/Ptnano catalyst displayed a higher performance than MWCNTs. Under the optimized conditions of applied potential 0.5 V, pH 6.4, room temperature, the proposed biosensor showed a large determination range (0.2–2.0 mM), a short response time (within 5 s), a high sensitivity (6.36 μA mM − 1 ) and good stability (90% remains after 4 weeks). The fabricated biosensor had practically good selectivity against interferences. The results for whole blood samples measured by the present biosensor showed a good agreement with those measured by spectrophotometric method.

A novel amperometric choline biosensor has been fabricated with choline oxidase (ChOx) immobilized by the sol-gel method on the surface of multi-walled carbon nanotubes (MWCNT) modified platinum electrode to improve the sensitivity and the anti-interferential property of the sensor. By analyzing the electrocatalytic activity of the modified electrode by MWCNT, it was found that MWCNT could not only improve the current response to H2O2 but also decrease the electrocatalytic potential. The effects of experimental variables such as the buffer solutions, pH and the amount of loading enzyme were investigated for the optimum analytical performance. This sensor shows sensitive determination of choline with a linear range from 5.0 × 10−6 to 1.0 × 10−4 mol/L when the operating pH and potential are 7.2 and 0.15 V, respectively. The detection limit of choline was 5.0 × 10−7 mol/L. Selectivity for choline was 9.48 μA·(mmol/L)−1. The biosensor exhibits excellent anti-interferential property and good stability, retaining 85% of its original current value even after a month. It has been applied to the determination of choline in human serum.

A new type of amperometric l -lactate biosensor based on silica sol-gel and multi-walled carbon nanotubes (MWCNTs) organic–inorganic hybrid composite material was developed. The sol-gel film was used to immobilize l -lactate oxidase on the surface of glassy carbon electrode (GCE). MWCNTs were used to increase the current response and improve the performance of biosensor. The sol-gel film fabrication process parameters such as H 2 O : TEOS and pH were optimized, Effects of some experimental variables such as applied potential, temperature, and pH on the current response of the biosensor were investigated. Analytical characteristics and dynamic parameters of the biosensors with and without MWCNTs in the hybrid film were compared, and the results showed that analytical performance of the biosensor could be improved greatly after introduction of the MWCNTs. Sensitivity, linear range, limit of detection ( S / N = 3) were 2.097 μA mM − 1 , 0.3 to 1.5 mM, 0.8 × 10 − 3 mM for the biosensor without MWCNTs and 6.031 μA mM − 1 , 0.2 to 2.0 mM, 0.3 × 10 − 3 mM for the biosensor with MWCNTs, respectively. This method has been used to determine the l -lactate concentration in real human blood samples.

In this work, a simple, label-free, low cost electrochemical biosensor for highly sensitive and selective detection of Escherichia coli has been developed on the basis of rolling circle amplification (RCA) coupled peroxidase-mimicking DNAzyme amplification. A aptamer-primer probe (APP) containing anti-E. coli aptamer and a primer sequence complementary to a circular probe, which includes two G-quadruplex units, is used for recognizing target and triggering RCA-based polymerase elongation. Due to RCA coupled DNAzyme amplification strategy, the presence of target E. coli leads to the formation of numerous G-quadruplex oligomers on electrode, which folds into G-quadruplex/hemin complexs with the help of K(+) and hemin, thus generating extremely strong catalytic activity toward H2O2 and giving a remarkably strong electrochemical response. As far as we know, this work is the first time that RCA coupled peroxidase-mimicking DNAzyme amplification technique have been integrated into electrochemical assay for detecting pathogenic bacteria. Under optimal conditions, the proposed biosensor exhibits ultrahigh sensitivity toward E. coli with detection limits of 8cfumL(-1) and a detection range of 5 orders of magnitude. Besides, our biosensor also shows high selectivity toward target E. coli and has the advantages in its rapidness, low cost, simplified operations without the need of electrochemical labeling steps and additional labile reagents. Hence, the RCA coupled peroxidase-mimicking DNAzyme amplification-based electrochemical method might create a useful and practical platform for detecting E. coli and related food safety analysis and clinical diagnosis.

A low-cost, simple, portable and sensitive paper-based electrochemical sensor was established for the detection of K-562 cell in point-of-care testing. The hybrid material of 3D Au nanoparticles/graphene (3D Au NPs/GN) with high specific surface area and ionic liquid (IL) with widened electrochemical windows improved the good biocompatibility and high conductivity was modified on paper working electrode (PWE) by the classic assembly method and then employed as the sensing surface. IL could not only enhance the electron transfer ability but also provide sensing recognition interface for the conjugation of Con A with cells, with the cell capture efficiency and the sensitivity of biosensor strengthened simultaneously. Concanavalin A (Con A) immobilization matrix was used to capture cells. As proof-of-concept, the paper-based electrochemical sensor for the detection of K-562 cells was developed. With such sandwich-type assay format, K-562 cells as model cells were captured on the surface of Con A/IL/3D AuNPs@GN/PWE. Con A-labeled dendritic PdAg NPs were captured on the surface of K-562 cells. Such dendritic PdAg NPs worked as catalysts promoting the oxidation of thionine (TH) by H2O2 which was released from K-562 cells via the stimulation of phorbol 12-myristate-13-acetate (PMA). Therefore, the current signal response was dependent on the amount of PdAg NPs and the concentration of H2O2, the latter of which corresponded with the releasing amount from cells. So, the detection method of K-562 cell was also developed. Under optimized experimental conditions, 1.5×10(-14) mol of H2O2 releasing from each cell was calculated. The linear range and the detection limit for K-562 cells were determined to be 1.0×10(3)-5.0×10(6) cells/mL and 200 cells/mL, respectively. Such as-prepared sensor showed excellent analytical performance with good fabrication reproducibility, acceptable precision and satisfied accuracy, providing a novel protocol in point-of-care testing of cells.

We report here the development of an optical biosensor based on the resonant mirror for kinetic analysis of soluble Interleukin-1 receptor I (sIL-1R I) in solution binding to immobilized Interleukin-1alpha (IL-1alpha). IL-1alpha was immobilized through its surface amine groups via amide bonds with the carboxyl groups of the carboxymethyl dextran (CMD) on cuvette surface. The interaction of sIL-1R I and IL-1alpha was monitored in real time. Evaluation of the binding curves allowed the analysis of the binding kinetics. The linear range of sIL-1R I in solution was over a range of 100-1600nM (R=0.9962). Equilibrium dissociation constant (K(D)) was derived by Scatchard plot analysis for sIL-1R I binding to immobilized IL-1alpha. For this assay, the K(D) was 2.6x10(-6)M. The CMD cuvette modified by IL-1alpha was successfully regenerated using 10mM HCl, and the same sensing surface was used repeatedly for the interaction analysis.

Paeoniae radix 801 is one of the active ingredients of the blood-activating and stasis-eliminating traditional Chinese medicine (TCM). In order to find out its action mechanism, an interaction assay system (IAsys) biosensor and a quartz crystal microbalance (QCM) were employed for the study on the specific interaction between P. radix 801 and endothelin-1 (ET-1). In the experiments, ET-1 was immobilized on the surfaces of the IAsys biosensor cuvettes and the QCM substrates by surface modification techniques, and then P. radix 801 was added to the cuvette and the substrate, separately. Then, the binding and interaction process between P. radix 801 and ET-1 was monitored. The results showed that P. radix 801 bound ET-1 specifically and the average binding masses were 1.28 ng mm−2 on the IAsys biosensor and 135 ng cm−2 (=1.35 ng mm−2) on the QCM sensor, respectively. Therefore, P. radix 801 generates bioactivity by binding and restraining the activity of ET-1. Both the IAsys biosensor and the QCM measurement techniques had high reproducibility and reliability and were found to be reliable tools to study the interaction of P. radix 801 and ET-1. This finding offers a new and effective way to study the action mechanism of P. radix 801.

The loading of multi-walled carbon nanotubes (MWNTs) and glucose oxidase (GOx) in the alternate layers of a glucose biosensor was first optimized based on a layer-by-layer construction on the surface of a graphite disk electrode. With the increasing of MWNTs/GOx layers, the response current to glucose was changed regularly and the response current reached a maximum value when the number of MWNTs/GOx layers was 6. Owing to a good electrical conductivity, strong adsorption and excellent bioconsistency of MWNTs, the (MWNTs/GOx) 6 films-coated glucose biosensor had an excellent electrochemical properties. The response current of the (MWNTs/GOx) 6 films-coated biosensor to 3 × 10 − 2 M glucose was 1.63 μA while the response time was only 6.7 s. The linear range and the lowest detectable concentration of this biosensor was 5 × 10 − 4 ∼1.5 × 10 − 2 M and 0.9 × 10 − 4 M, respectively.

A novel method for real time kinetic analysis of the interaction between IL-1α and sIL-1R I is reported. sIL-1R I was immobilized on the biotin cuvette surface of the resonant mirror biosensor to set up the experimental model. Results obtained from the assay confirmed that biotinylated sIL-1R I was specifically immobilized on the avidin-coated biotin cuvette surface, the interaction between IL-1α and sIL-1R I was fast and specific, and the interaction response is dose-dependence of IL-1α in solution with a range of 150–2400 nM. The binding curve was fitted by FASTfit analysis, which fitted the monophasic association quite well, and the error did not exceed 0.4 arc seconds. Kinetic constants for IL-1α binding to sIL-1R I were determined from the plots of K on versus the concentration of IL-1α. For the interaction, k ass was 2.81 × 10 3 M −1 s −1 , K diss was 2.52 × 10 −3 s −1 , and K D was 8.97 × 10 −7 M.

A novel electrochemical immunosensor assay (EIA) for highly sensitive and specific detection of Escherichia coli O157:H7 has been developed. This immunosensor is constructed by the assembly of capture antibody on SG-PEDOT-AuNPs composites modified glass carbon electrode. In the presence of target E. coli O157:H7, horseradish peroxidase (HRP)-labeled antibody is captured on the electrode surface to form a sandwich-type system via the specific identification. As a result, E. coli O157:H7 detection is realized by outputting a redox current from electro-reduction of hydrogen peroxide reaction catalyzed by HRP. In our assay, the combination of the unique properties of sulfonated graphene (SG) and gold nanoparticles (AuNPs) can not only accelerate electron transfer on electrode interface, but also provide an excellent scaffold for the conjugation of capture antibody that significantly improves the target capture efficiency and enhances the sensitivity of the biosensor. The results reveal the calibration plot obtained for E. coli O157:H7 is approximately linear from 7.8 × 10-7.8 × 10(6) colony-forming unit (cfu) mL(-1) with the limit of detection of 3.4 × 10 cfu mL(-1). In addition, the biosensor has been successfully applied to the quantitative assay of E. coli O157:H7 in synthetic samples (spring water and milk). Hence, the developed electrochemical-based immunosensor might provide a useful and practical tool for E. coli O157:H7 determination and related food safety analysis and clinical diagnosis.

To study the effects of propyl gallate on the interaction of tumor necrosis factor-α (TNF-α) with its soluble receptor, sTNFR-I. Interactions between TNF-α and sTNFR-I were analyzed using an IAsys biosensor. sTNFR-I was immobilized on the carboxymethyl dextran (CMD) surface of the IAsys biosensor cuvettes, and TNF-α preincubated with different concentrations of propyl gallate was added to the cuvettes. The resonant angle shift caused by the binding between TNF-α and sTNFR-I was then recorded. sTNFR-I was immobilized on the CMD surface at a density of 2.76 ng/mm2. TNF-α then bound the immobilized sTNFR-I specifically, and propyl gallate was able to enhance the binding between TNF-α and sTNFR-I in a dose-dependent manner. The binding between TNF-α and sTNFR-I is one of the targets that propyl gallate can act on in vivo. The IAsys biosensor offers a new clue as to the study on the mechanisms of action of propyl gallate.

An acetylcholine (ACh) biosensor has been fabricated with bienzymes/poly(diallyldimethylammonium chloride) (PDDA) multilayer film-modified platinum (Pt) electrodes by a layer-by-layer technique (LBL). The ACh biosensor was optimized and the properties are described. This ACh biosensor was used for the detection of organophosphate pesticide trichlorfon. The detection limits (found 0.001 μg/mL for trichlorfon) make it possible to detect the pollutants. This simple protocol of biosensor preparation, high sensitivity and stability are very promising for the determination of environmental pollutants in field conditions.

A layer-by-layer deposition technique was employed for fabricating choline biosensors using chloline oxidase (ChOx) and polycations such as poly(ethyleneimine) (PEI) and poly(diallyldimethylammonium chloride) (PDDA). A platinum electrode was coated with ChOx/PEI or ChOx/PDDA thin film to prepare amperometric choline sensors. The amperometric response of the sensors depended significantly on the type of the polycations. The ChOx/PDDA film suppressed the permeation of choline, resulting in a lower response than that of the ChOx/PEI film-based sensors. The results were rationalized based on the different chemical structures of the polycationic materials.

A resonant mirror biosensor, IAsys, and a quartz crystal microbalance (QCM) are known independently as surface sensitive analytical devices capable of label-free and in situ bioassays. In this study, an IAsys and a QCM are employed for a new study on the action mechanism of Paeoniae Radix 801 (P. radix 801) by detecting the specific interaction between P. radix 801 and endothelin-1 (ET-1). In the experiments, ET-1 was immobilized on the surfaces of the IAsys cuvette and the QCM substrate by surface modification techniques, and then P. radix 801 solution was contacted to the cuvette and the substrate, separately. Then, the binding and interaction process between P. radix 801 and ET-1 was monitored by IAsys and QCM, respectively. The experimental results showed that P. radix 801 binds ET-1 specifically. The IAsys and QCM response curves to the ET-1 immobilization and P. radix 801 binding are similar in reaction process, but different in binding profiles, reflecting different resonation principles. Although both IAsys and QCM could detect the interaction of P. radix 801 and ET-1 with high reproducibility and reliability through optimization of the ET-1 coating, the reproducibility and reliability obtained by IAsys are better than those obtained by QCM, since the QCM frequency is more sensitive to temperature fluctuations, atmospheric changes and mechanical disturbances. However, IAsys and QCM are generally potent and reliable tools to study the interaction of P. radix 801 and ET-1, and can conclusively be applied to the action mechanism of P. radix 801.

A new strategy for fabricating glucose biosensor was presented by layer-by-layer assembled chitosan (CS)/gold nanoparticles (GNp)/glucose oxidase (GOD) multilayer films modified Pt electrode. First, a cleaned Pt electrode was immersed in poly(allylamine) (PAA), and then transferred to GNp, followed by the adsorption of GOD (GOD/GNp/PAA/Pt). Second, the GOD/GNp/PAA/Pt electrode was immersed in CS, and then transferred to GNp, followed by the adsorption of GOD (GOD/GNp/CS/GOD/GNp/PAA/Pt). Third, different layers of multilayer films modified Pt electrodes were assembled by repeating the second process. Film assembling and characterization were studied by quart crystal microbalance, and properties of the resulting glucose biosensors were measured by electrochemical measurements. The results confirmed that the assembling process of multilayer films was simple to operate, the immobilized GOD displayed an excellent catalytic property to glucose, and GNp in the biosensing interface efficiently improved the electron transfer between analyte and electrode surface. The amperometric response of the biosensors uniformly increased from one to six layers of multilayer films, and then reached saturation after the seven layers. Among the resulting biosensors, the biosensor based on the six layers of multilayer films was best. It showed a wide linear range of 0.5-16 mM, with a detection limit of 7.0 microM estimated at a signal-to-noise ratio of 3, fast response time (within 8s). Moreover, it exhibited good reproducibility, long-term stability and interference free. This method can be used for constructing other thin films, which is a universal immobilization method for biosensor fabrication.

An amperometric choline biosensor was developed by immobilizing choline oxidase (ChOx) in a layer-by-layer (LBL) multilayer film on a platinum (Pt) electrode modified with Prussian blue (PB). 6-O-Ethoxytrimethylammoniochitosan chloride (EACC) was used to prepare the ChOx LBL films. The choline biosensor was used at 0.0 V versus Ag/AgCl to detect choline and exhibited good characteristics such as relative low detection limit (5 × 10−7 M), short response time (within 10 s), high sensitivity (88.6 μA mM−1 cm−2) and a good selectivity. The results were explained based on the ultrathin nature of the LBL films and the low operating potential that could be due to the efficient catalytic reduction of H2O2 by PB. In addition, the effects of pH, temperature and applied potential on the amperometric response of choline biosensor were evaluated. The apparent Michaelis–Menten constant was found to be (0.083 ± 0.001) ×10−3 M. The biosensor showed excellent long-term storage stability, which originates from a strong adsorption of ChOx in the EACC multilayer film. When the present choline biosensor was applied to the analysis of phosphatidylcholine in serum samples, the measurement values agreed satisfactorily with those by a hospital method.

The sequence-specific detection of DNA hybridization has attracted considerable interest in numerous fields. Although traditional DNA biosensors have been widely explored due to their high sensitivity, it is still challenging to develop a low-cost, portable, disposable, fast, and easy-to-use DNA detection method for public use at home or in the field. To address these challenges, herein, we report a novel microfluidic photoelectrochemical (PEC) paper-based analytical platform, integrated with an internal chemiluminescent light source, a novel paper supercapacitor (PS) amplifier, and a terminal digital multi-meter (DMM) detector, for sensitive DNA detection using a graphene-modified porous Au-paper electrode as the working electrode to obtain enhanced PEC responses. The quantification mechanism of this strategy is based on the charging of this PS, which was constructed on a paper-based analytical platform through a simple "drawing and soaking" method, by the generated photocurrent. After a fixed period, the PS was automatically shorted under the control of a novel built-in fluidic delay-switch to output an instantaneously amplified current, which could be sensitively detected by the DMM. At optimal conditions, this paper-based analytical platform can detect DNA at concentrations at femtomolar level. This approach also shows excellent specificity toward single nucleotide mismatches.

A sensitive electrochemiluminescence (ECL) DNA biosensor based on nanoporous gold (NPG) electrode and PdCu@carbon nanocrystals (CNCs) composites is developed. The CNCs were obtained simply by electrooxidation with abundant carboxyl groups at their surfaces. The NPG can be easily prepared by a selective dissolution of silver from silver-gold alloy in nitric acid, which has free-standing noble metal membranes with controllable three-dimensional (3D) porosity. The PdCu bimetallic nanocomposites with hierarchically hollow structures were fabricated through a simple replacement reaction using dealloyed nanoporous copper (NPC) as both a template and reducing agent. Structure characterization was obtained by means of transmission electron microscope (TEM) and scanning electron microscope (SEM) images. The PdCu@CNCs composites exhibit 6 times higher ECL intensity than the pure CNC-labeled reporter DNA. Taking advantage of dual-amplification effects of the developed probe, a limit of detection as low as 18 aM can be achieved and the assay exhibits excellent selectivity for single-mismatched DNA detection even in human serum. The proposed ECL based method should have wide applications in diagnosis of genetic diseases due to its simplicity, low cost, and high sensitivity at extremely low concentrations.

In this study, a novel microfluidic paper-based chemiluminescence analytical device (μPCAD) with a simultaneous, rapid, sensitive and quantitative response for glucose and uric acid was designed. This novel lab-on-paper biosensor is based on oxidase enzyme reactions (glucose oxidase and urate oxidase, respectively) and the chemiluminescence reaction between a rhodanine derivative and generated hydrogen peroxide in an acid medium. The possible chemiluminescence assay principle of this μPCAD is explained. We found that the simultaneous determination of glucose and uric acid could be achieved by differing the distances that the glucose and uric acid samples traveled. This lab-on-paper biosensor could provide reproducible results upon storage at 4 °C for at least 10 weeks. The application test of our μPCAD was then successfully performed with the simultaneous determination of glucose and uric acid in artificial urine. This study shows the successful integration of the μPCAD and the chemiluminescence method will be an easy-to-use, inexpensive, and portable alternative for point-of-care monitoring.