Isothermal strand displacement polymerase reaction (ISDPR)-assisted microchip electrophoresis for highly sensitive detection of cancer associated microRNAs.

More and more studies have found that microRNAs (miRNAs) are markers of cancer, and detection of miRNAs is beneficial for early diagnosis and prognosis of cancer. In this paper, the isothermal strand displacement polymerase reaction (ISDPR), which is an enzyme-assisted nucleic acid amplification method, was studied to combine with microchip electrophoresis (MCE) for a simultaneously detection of two cancer related miRNAs named microRNA-21 (miR-21) and microRNA-221 (miR-221). In the ISDPR amplification, two different DNA hairpins (HPs) were specifically designed, so that miR-21 and miR-221 could respectively bind to HPs and started ISDPR amplification to generate two different products which were ultimately detected by MCE. The optimal conditions of ISDPR were carefully investigated, and the limits of detection (LOD) of miR-21 and miR-221 were as low as 0.35 fM and 0.25 fM (S/N = 3) respectively under these conditions. The human lung tumor cells and serum samples were analyzed by this ISDPR-MCE method and satisfactory results were obtained, which means that this method is of high sensitivity, high efficiency, low reagent consumption and simple operation in miRNAs detection.

Ultrasensitive rapid detection of antibiotic resistance genes by electrochemical ratiometric genosensor based on 2D monolayer Ti3C2@AuNPs.

As an important emerging pollutant, antibiotic resistance genes (ARGs) monitoring is crucial to protect the ecological environment and public health, but its rapid and accurate detection is still a major challenge. In this study, a new single-labeled dual-signal output ratiometric electrochemical genosensor (E-DNA) was developed for the rapid and highly sensitive detection of ARGs using a synergistic signal amplification strategy of T3C2@Au nanoparticles (T3C2@AuNPs) and isothermal strand displacement polymerase reaction (ISDPR). Specially, two-dimensional monolayer T3C2 nanosheets loaded with uniformly gold nanoparticles were prepared and used as the sensing platform of the E-DNA sensor. Benefiting from excellent conductivity and large specific surface area of Ti3C2@AuNPs, the probe immobilization capacity of the E-DNA sensor is doubled, and electrochemical response signals of the E-DNA sensor were significantly improved. The proposed single-labeled dual-signal output ratiometric sensing strategy exhibits three to six times higher sensitivity for the sul2 gene than the single-signal sensing strategy, which significantly reduces cost meanwhile retaining the advantages of high sensitivity and reliability offered by conventional dual-labeled ratiometric sensors. Coupled with ISDPR amplification technology, the E-DNA sensor has a wider linear range from 10 fM to 10 nM and a limit of detection as low as 2.04 fM (S/N=3). More importantly, the E-DNA sensor demonstrates excellent specificity, good stability and reproducibility for target ARGs detection in real water samples. The proposed new sensing strategy provides a highly sensitive and versatile tool for the rapid and accurate quantitative analysis of various ARGs in environmental water samples.

Ultrasensitive microchip electrophoretic detection of the mecA gene in methicillin-resistant Staphylococcus aureus (MRSA) based on isothermal strand-displacement polymerase reaction.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the main pathogens involved in hospital and community infection. To rapidly and sensitively detect the mecA gene, which is relevant to methicillin-resistant strains, microchip electrophoresis (MCE) integrated with isothermal strand-displacement polymerase reaction (ISDPR) was developed. In the ISDPR signal recycle amplification, the target DNA opened the DNA hairpin structure by specifically binding with the hairpin probe (HP), and then the primer hybridized with the probe and released the target DNA in the presence of Klenow Fragment exo- (KF exo-) polymerase. The released target DNA hybridized with the next HP and then was displaced by the primer again, consequently achieving target recycling and amplification. The amplified products of the HP-cDNA duplex were separated rapidly from other DNAs by MCE. Under optimal conditions, the limit of detection of the target DNA was as low as 12.3 pM (S/N = 3). The proposed ISDPR with MCE method was also successfully applied to detect methicillin-resistant S. aureus, and the experimental results showed that it had some advantages such as being label free, ultrasensitive, rapid and well separated.

Signal-on electrogenerated chemiluminescence biosensor for ultrasensitive detection of microRNA-21 based on isothermal strand-displacement polymerase reaction and bridge DNA-gold nanoparticles.

MicroRNA-21 (miRNA-21) is a promising diagnostic biomarker for breast cancer screening and disease progression. A sensitive and selective strategy for the quantitative determination of miRNA-21 is of great significance in the early diagnosis of cancers. Herein, a novel electrogenerated chemiluminescence (ECL) biosensor was designed for the detection of miRNA-21 with dual signal amplification based on isothermal strand-displacement polymerase reaction (ISDPR) and bridge DNA-gold nanoparticles (AuNPs) nanocomposites. The ECL biosensor was fabricated by self-assembling a thiolated capture probe (SH-CP) on the surface of a gold electrode. The target miRNA-21 initiated the phi29 DNA polymerase-mediated ISDPR, which could generate large numbers of single-stranded DNA (assistant DNA) with accurate and comprehensive nucleotide sequences. The assistant DNA was captured by the SH-CP self-assembled on the Au electrode and further hybridized with bridge DNA-AuNPs nanocomposites, more biotins can be captured on the electrode surface. Afterward, a streptavidin-modified Ru (bpy)32+ complex (SA-Ru) was bound to the bridge DNA-AuNPs nanocomposites via a specific interaction between biotin and streptavidin to produce a strong ECL signal. The ECL intensity was logarithmically proportion to the concentration of target miRNA-21 over a range from 0.01 fM to 10,000 fM with a detection limit of 3.2 aM. The proposed ECL biosensor was successfully applied to detect miRNA-21 in total RNA samples extracted from human breast cancer cells, and it showed great potential for early cancer diagnosis based on miRNA as a biomarker.

Multipedal DNA Walker Biosensors Based on Catalyzed Hairpin Assembly and Isothermal Strand-Displacement Polymerase Reaction for the Chemiluminescent Detection of Proteins.

In this study, two kinds of sensitive biosensors based on a multipedal DNA walker along a three-dimensional DNA functional magnet particles track for the chemiluminescent detection of streptavidin (SA) are constructed and compared. In the presence of SA, a multipedal DNA walker was constructed by a biotin-modified catalyst as a result of the terminal protection to avoid being digested by exonuclease I. Then, through a toehold-mediated strand exchange, a "leg" of a multipedal DNA walker interacted with a toehold of a catalyzed hairpin assembly (CHA)-H1 coupled with magnetic microparticles (MMPs) and opened its hairpin structure. The newly open stem in CHA-H1 was hybridized with a toehold of biotin-labeled H2. Via the strand displacement process, H2 displaced one "leg" of a multipedal DNA walker, and the other "leg" continued to interact with the neighboring H1 to initiate the next cycle. In order to solve the high background caused by the hybridization between CHA-H1 and H2 without a CHA-catalyst, the other model was designed. The principle of the other model (isothermal strand-displacement polymerase reaction (ISDPR)-DNA walker) was similar to that of the above one. After the terminal protection of SA, a "leg" of a multipedal DNA walker was triggered to open the hairpin of the ISDPR-H1 conjugated with MMPs. Then, the biotin-modified primer hybridized with the newly exposed DNA segment, triggering the polymerization reaction with the assistance of dNTPs/polymerase. As for the extension of the primer, the "leg" of a multipedal DNA walker was displaced so that the other "leg" could trigger the proximal H1 to go onto the next cycle. Due to its lower background and stronger signal, a multipedal DNA walker based on an ISDPR had a lower limit of detection for SA. The limit of detection for SA was 6.5 pM, and for expanding the application of the method, the detections of the folate receptor and thrombin were explored. In addition, these DNA walker methods were applied in complex samples successfully.

Photoelectrochemical biosensor for microRNA detection based on multiple amplification strategies.

A photoelectrochemical biosensor is described for sensitive detection of microRNA-162a. A multiple amplification strategy is employed that involves (a) isothermal strand displacement polymerase reaction; (b) terminal deoxynucleotidyl transferase-mediated extension, (c) amplification of streptavidin-coated gold nanoparticles, and (d) biotin functionalized alkaline phosphatase. Graphite-like C3N4 (g-C3N4) nanosheets were used as photoactive material. By using these amplification strategies, the detection limit is as low as 0.18 fM of microRNA, and the photocurrent increases linearly with the concentration of microRNA-162a in the range from 0.5 fM to 1 pM. The method was successfully applied to quantify the expression level of microRNA-162a in total RNA extracted from the leaves of maize seedlings after incubation with the chemical mutagen ethyl methanesulfonate. The results confirmed the applicability of the method to the analysis of practical biological samples. Graphical Abstract Schematic of a photoelectrochemical microRNA assay based on a multiple amplification strategy involving (a) isothermal strand displacement polymerase reaction; (b) terminal deoxynucleotidyl transferase-mediated extension,

Signal-on electrochemiluminescence biosensor for microRNA-319a detection based on two-stage isothermal strand-displacement polymerase reaction.

MicroRNAs play crucial role in regulating gene expression in organism, thus it is very necessary to exploit an efficient method for the sensitive and specific detection of microRNA. Herein, a signal-on electrochemiluminescence biosensor was fabricated for microRNA-319a detection based on two-stage isothermal strand-displacement polymerase reaction (ISDPR). In the presence of target microRNA, amounts of trigger DNA could be generated by the first ISDPR. Then, the trigger DNA and the primer hybridized simultaneously with the hairpin probe to open the stem of the probe, and then the ECL signal will be emitted. In the presence of phi29 DNA polymerase and dNTPs, the trigger DNA could be displaced to initiate a new cycle which was the second ISDPR. Due to the two-stage amplification, this method presented excellent detection sensitivity with a low detection limit of 0.14 fM. Moreover, the applicability of the developed method was demonstrated by detecting the change of microRNA-319a content in the leaves of rice seedlings after the rice seeds were incubated with chemical mutagen of ethyl methanesulfonate.

A graphene oxide-based strand displacement amplification platform for ricin detection using aptamer as recognition element.

The toxic plant protein ricin is a potential agent for criminal or bioterrorist attacks due to the wide availability and relative ease of preparation. Herein, we developed a novel strategy for the detection of ricin B-chain (RTB) based on isothermal strand-displacement polymerase reaction (ISDPR) by using aptamer as a recognition element and graphene oxide (GO) as a low background platform. In this method, ricin-binding aptamer (RBA) hybridized with a short blocker firstly, and then was immobilized on the surface of streptavidin-coated magnetic beads (MBs). The addition of RTB could release the blocker, which could hybridize with the dye-modified hairpin probe and trigger the ISDPR, resulting in high fluorescence intensity. In the absence of RTB, however, the fluorescence of the dye could be quenched strongly by GO, resulting in the extremely low background signal. Thus, RTB could be sensitively detected by the significantly increased fluorescence signal. The linear range of the current analytical system was from 0.75µg/mL to 100µg/mL and the limit of detection (3σ) was 0.6µg/mL. This method has been successfully utilized for the detection of both the RTB and the entire ricin toxin in real samples, and it could be generalized to any kind of target detection based on an appropriate aptamer.