Signal Amplification Strategy Research Articles

MXene@MnIn2S4‐Gated Organic Photoelectrochemical Transistors with Nanozyme‐Mediated Multiple Quenching Effects for Ultrasensitive Detection of Okadaic Acid

AbstractOrganic optoelectronics have attracted widespread interdisciplinary research interest but lags far behind in the application in marine environmental detection. The organic photoelectrochemical transistor (OPECT) shows promise as a powerful tool for comprehensive monitoring and early warning of marine conditions, which can be further enhanced by the valuable signal amplification strategy of nanozyme‐mediated catalytic precipitation. Herein, OPECT technology is integrated with nanozyme‐mediated catalytic precipitation for the first time, establishing an ultrasensitive detection platform for okadaic acid (OA). Specifically, MXene@MnIn2S4 (MXMnIS) hybrid composed of Schottky‐junction is synthesized via a hydrothermal method, which can efficiently modulate the device with high current gain. Linking with a sandwich immunoassay, the Ru‐C3N4 nanozyme with peroxidase‐mimicking activity can catalyze the oxidation of 4‐chloro‐1‐naphthol (4‐CN) to form an insoluble precipitate on the electrode surface, resulting in a decrease in the photocurrent and altering the transistor response. Importantly, the proposed OPECT biosensor presented an excellent sensitivity and a low detection limit (32.5 pM), fully satisfying the fundamental requirements for the quantitative detection of intracellular and extracellular OA in the practical culture media of Prorocentrum lima at different growth stages. This OPECT platform based on the nanozyme‐mediated quenching effect is significant for effectively monitoring the safety of the marine ecological environment and food safety.

Dual-sensitized heterojunction Ag2S/ZnS/NiS composites with entire visible-light region absorption for ultrasensitive photoelectrochemical detection of tobramycin

In this study, an ultrasensitive photoelectrochemical (PEC) aptasensor based on dual-sensitized heterojunction Ag2S/ZnS/NiS composites as a signal probe was proposed for the detection of tobramycin (TOB) by combining a cascaded quadratic signal amplification strategy. Specifically, compared to the limited visible light-harvesting capability of single sensitized composites, Ag2S/ZnS/NiS composites with p-n and n-n heterojunction could greatly improve the light energy utilization to tremendously strengthen the optical absorption in the entire visible-light region. Moreover, dual-sensitized heterojunction could effectively hinder the rapid recombination of photoelectrons and holes (carriers) to obtain a good photocurrent for improving the sensitivity of the aptasensor. Furthermore, a cascaded quadratic signal amplification strategy was applied to convert trace target TOB into plentiful gold nanoclusters (Au NCs) labelled double-stranded DNA for the construction of PEC aptasensor, with a broad linear detection range from 0.01 to 100 ng mL−1 and a low detection limit of 3.38 pg mL−1. Importantly, this study provided a versatile and sensitive PEC biosensing platform for TOB analysis, and demonstrated its successful application for TOB detection in milk samples. This protocol provides a novel dual-sensitized heterojunction composites to develop a highly efficient and harmfulless PEC aptasensor, which is expected to be used in food safety, environmental monitoring and other areas.

A covalent organic framework nanosheet-nanochannel composite with signal amplification strategy for electrochemical enantioselective recognition

Recognition and separation of chiral isomers are of great importance in both industrial and biological applications. However, owing to identical molecular formulas and chemical properties of enantiomers, signal transduction and amplification are still two major challenges in chiral sensing. In this study, we developed an enantioselective device by integrating chiral covalent organic framework nanosheets (CONs) with nanochannels for sensitive identification and quantification of enantiomers. Using 3,4-dihydroxyphenylalanine (DOPA) as the model analyte, the as-prepared chiral nanofluidic device exhibits a remarkable chiral recognition ability to l-DOPA than d-DOPA. More importantly, due to the chelation of DOPA with Fe3+ ions, it can efficiently block the ion transport through channel and shield the channel surface charge, which will amplify the difference in the electrochemical response of l-DOPA and d-DOPA. Therefore, a sensitive chiral recognition can be achieved using the present nanofluidic device coupled using electrochemical amplification strategy. Notably, using this method, an ultra-low concentration of l-DOPA (as low as 0.21 pM) can be facilely and successfully detected with a linear range of 1 pM–10 μM. This study provides a reliable and sensitive approach for achieving highly selective detection of chiral molecules.

Peptide-based electrochemical detection of prostate cancer-derived exosomes using a dual signal amplification strategy

Prostate cancer-derived exosomes have important potential as biomarkers for diagnosis and treatment of prostate cancer. But such specific exosomes towards clinical application remains problematic due to their comparatively low concentration in relation to other constituents of blood. Additionally, the presence of particles in blood that share a similar size with exosomes adds to the complexity of their selective and sensitive detection. Consequently, the detection of exosomes derived from prostate cancer in intricate biological settings necessitates the implementation of highly sensitive and specific biosensors. Herein, we report an electrochemical biosensor for prostate cancer-derived exosomes detection, with two-level selectivity achieved through a sandwich structure involving specific peptides and two-level amplification utilizing the combination of biotin-streptavidin linkage and G-quadruplex hemin mimetic peroxidase to enhance the sensitivity. Evaluation of PSMA positive exosomes at various concentrations demonstrates a remarkable limit of detection as low as 26 particles/μL, as well as an excellent linear sensor response spanning from 1.0 × 102 to 1.0 × 107 particles/μL. Compared to the enzymatic biosensor, this biosensor proves more versatile without a label or enzyme, and may be more promising for clinical applications.

Highly sensitive electrochemical quantification of carbendazim via synergistic enhancement of ring-opening metathesis polymerization and polyethyleneimine modified graphene oxide.

Anovel aptamer-based sensor was developed using the signal amplification strategy of ring-opening metathesis polymerization (ROMP) and polyethyleneimine modified graphene oxide to achieve trace detection of carbendazim (CBZ). The dual identification of aptamer and antibody was used to avoid false positive results and improve the selectivity. Polyethyleneimine modified graphene oxide (GO-PEI), as a substrate material with excellent conductivity, was modified on the surface of aglassy carbon electrode (GCE) to increase the grafting amount of aptamer on the electrode surface. Moreover, a large number of cyclopentenyl ferrocene (CFc) was aggregated to form long polymer chains through ring-opening metathesis polymerization (ROMP), so as to significantly improve the detection sensitivity of the biosensor. The linear range of this sensor was 1 pg/mL-100 ng/mL with a detection limit as low as 7.80fg/mL. The sensorexhibited excellent reproducibility and stability, and also achieved satisfactory results in actual sample detection. The design principle of such a sensor could provide innovative ideas for sensors in the detection of other types of targets.

Dual signal amplification strategy for enhancing Eu2Sn2O7 electrochemiluminescence immunosensor for the sensitive detection of N-terminal pro-brain natriuretic peptide

Pyrochlore oxide Eu2Sn2O7, an electrochemiluminescence (ECL) emitter, exhibits favorable characteristics such as low toxicity, good stability, and abundant electron vacancies. In this work, the interfacial electron transfer efficiency of Eu2Sn2O7 was significantly enhanced through the in situ surface modification with silver nanoparticles (AgNP). Moreover, we introduced a dual signal-amplification strategy involving AgNP and H2O2. The AgNP as a co-reaction catalyst facilitated the reduction of the co-reactant K2S2O8. The OH• generated through H2O2 reduction on the electrode surface induced the decomposition of K2S2O8 to SO4•−. The MoS2-PEI-AuNP (PEI: polyethylenimine) composite was selected as an ideal substrate. A highly sensitive ECL immunosensor based on Eu2Sn2O7-Ag and MoS2-PEI-AuNP was constructed to detect N-terminal pro-brain natriuretic peptide (NT-proBNP). Under the optimized experimental conditions, the ECL immunosensor demonstrated a linear range of 10 fg·mL−1–50 ng·mL−1, with a detection limit of 2.9 fg·mL−1, and showed excellent stability and specificity. The results demonstrate the potential of the ECL immunosensor for clinical diagnosis applications.

Well-ordered Au@Ag NBPs/SiO2 nanoarray for sensitive detection of chloramphenicol via DNAzyme-assisted SERS sensing

Misuse of chloramphenicol (CAP) can lead to severe food safety issues. Therefore, the accurate and sensitive detection of CAP residues is important for public health. Herein, a convenient and reliable interfacial self-assembly technique was used to form a uniform Au@Ag nanobipyramids (NBPs) film on an ordered SiO2 nanosphere array (SiO2 NS), which served as a Raman-enhanced substrate. In conjunction with a deoxyribonucleic acid enzyme–induced signal amplification strategy, we developed a novel surface-enhanced Raman scattering (SERS) biosensor for the selective and sensitive detection of CAP. The biosensor exhibited a detection limit of 6.42 × 10−13 mol·L−1 and a detection range of 1.0 × 10−12–1.0 × 10−6 mol·L−1. The biosensor could detect CAP in spiked milk samples with a high accuracy, and its recovery rates ranged from 97.88% to 107.86%. The as-developed biosensor with the advantages of high sensitivity and high selectivity offers a new strategy for the rapid, reliable and sensitive detection of CAP, rendering it applicable to food safety control.

Development of folding-based and signal-amplified fluorescence aptasensors using G-quadruplex quinclorac aptamer variants engineered via exonuclease digestion and circular dichroism spectroscopy

The lack of inherent structure-switching functionality and inadequate affinity in aptamers hinders the construction and application of aptasensors. While exonuclease digestion-based strategies have been employed to engineer small-molecule aptamers with the structure-switching functionality and construct dual-exonuclease (exonuclease I and exonuclease III) digestion-based aptasensors, such studies targeting G-quadruplex (G4) aptamers remain scarce. This study focused on the exonuclease digestion and circular dichroism spectroscopy analysis of a G4 quinclorac (QNC) aptamer, Qapt-51, intending to design a functionalized QNC aptamer and a universal signal amplification strategy for constructing folding-based and sensitive dual-exonuclease digestion-based aptasensors, respectively. The exonuclease digestion results demonstrated that QNC-binding Qapt-51 inhibited the dual-exonuclease digestion. Combining the results from the circular dichroism spectroscopy, we further proposed a digestion mechanism for Qapt-51 involving a G4 conformational switch. Inspired by this mechanism, a functionalized Qapt-51 variant (Qapt-45A) was rationally designed to construct a simple and rapid folding-based fluorescence aptasensor. Additionally, we developed an exonuclease III- and phosphorodiamidate morpholino oligomer-assisted fluorescence signal amplification strategy, which can work in the dual-exonuclease digestion system. Leveraging this strategy integrated with the dual-exonuclease digestion of another Qapt-51 variant (AQ), a homogeneous signal-amplified fluorescence aptasensor was constructed to sensitively detect QNC without requiring the functionalized aptamers. These two aptasensors achieved detection limits of 560 ng/mL and 8.5 ng/mL for QNC. They successfully detected QNC in rice and river water samples with recoveries of 108.6%-87.5%. We hope that the successful development of these two aptasensors can provide valuable insights and references for the broader construction of G4 aptamer-based sensing systems.

Organic small molecule catalytic PET-RAFT electrochemical signal amplification strategy for miRNA-21 detection

Quantitative detection of the cancer-associated biomarker miRNA-21 is essential for the prevention and treatment of early-stage cancer patients. In the early diagnosis of cancer, due to the low concentration of miRNA in the organism, it is necessary to develop a fast, simple, highly sensitive and specific method using signal amplification techniques. Herein, a photo-induced electron/energy transfer reversible addition-fragmentation chain transfer polymerization signal amplification electrochemical biosensor was developed. Peptide nucleic acid was used as the recognition probe for miRNA-21 and the chain transfer agent (4-cyano-4-(dodecylsulfonylsulfanylthiocarbonyl)sulfonylpentanoic acid) bearing a carboxylic acid group was successfully conjugated with a phosphate group in the presence of ZrOCl2 to form the phosphate-Zr(IV)-carboxylic acid complex. Subsequently, the polymerization of the monomer (ferrocenylmethyl methacrylate) was achieved by blue light irradiation in the presence of erythrosin B (higher triple quantum yield) and triethanolamine (increases electron transfer rate). With this sophisticated signal amplification biosensing platform, low detection limit of 12.4 aM was achieved for miRNA-21. The biosensor reduces costs and simplifies experimentation while ensuring high sensitivity and selectivity. Meanwhile, the biosensor exhibits excellent anti-interference ability in serum samples and has great potential for practical applications in the early prevention of cancer.

Detection of fucosylated extracellular vesicles miR-4732-5p related to diagnosis of early lung adenocarcinoma by the electrochemical biosensor

Our preliminary investigation has identified the potential of serum fucosylated extracellular vesicles (EVs) miR-4732-5p in the early diagnosis of lung adenocarcinoma (LUAD) by a fucose-captured strategy utilizing lentil lectin (LCA)-magnetic beads and subsequent screening of high throughput sequencing and validation of real-time quantitative polymerase chain reaction (RT-qPCR). Considering the relatively complicated procedure, expensive equipment, and stringent laboratory condition, we have constructed an electrochemical biosensor assay for the detection of miR-4732-5p. miR-4732-5p is extremely low in serum, down to the fM level, so it needs to be detected by highly sensitive electrochemical methods based on the Mg2+-dependent DNAzyme splitting nucleic acid lock (NAL) cycle and hybridization chain reaction (HCR) signal amplification. In this study, signal amplification is achieved through the dual amplification reactions using NAL cycle in combination with HCR. In addition, hybridized DNA strands bind to a large number of methylene blue (MB) molecules to enhance signaling. Based on the above strategy, we further enhance our signal amplification strategies to improve detection sensitivity and accuracy. The implementation of this assay proceeded as follows: initially, miR-4732-5p was combined with NAL, and then Mg2+-dependent DNAzyme splitted NAL to release auxiliary DNA (S1) strands, which were subsequently captured by the immobilized capture probe DNA (C1) strands on the electrode surface. Following this, abundant quantities of DNA1 (H1) and DNA2 (H2) tandems were generated by HCR, and S1 strands then hybridized with the H1 and H2 tandems through base complementary pairing. Finally, MB was bonded to the H1 and H2 tandems through π–π stacking interaction, leading to the generation of a signal current upon the detection of a potential capable of inducing a redox change of MB by the electrode. Furthermore, we evaluated the performance of our developed electrochemical biosensor assay. The results demonstrated that our assay is a reliable approach, characterized by its high sensitivity (with a detection limit of 2.6 × 10−17 M), excellent specificity, good accuracy, reproducibility, and stability. Additionally, it is cost-effective, requires simple operation, and is portable, making it suitable for the detection of serum fucosylated extracellular vesicles miR-4732-5p. Ultimately, this development has the potential to enhance the diagnostic efficiency for patients with early-stage LUAD.

Target recognition-initiated allosteric probe-based multiple signal amplification strategy for sensitive and direct Pseudomonas aeruginosa detection

Pseudomonas aeruginosa (P. aeruginosa), a kind of gram-negative pathogenic bacteria, are causative agents of severe infections, such as lower respiratory tract infections in children and cancers. Detecting low levels of P. aeruginosa in clinical samples in an easy-to-operate manner is highly desired but still poses a problem. Herein, we established a target recognition-initiated allosteric probe-based multiple signal amplification strategy for sensitive detection of P. aeruginosa in a wash-free way. This approach involves the allosteric probe’s accurate recognition and binding to target P. aeruginosa, leading to subsequent multiple-cycle amplification. Afterward, the amplified products were translated to induce the aggregation of gold nanoparticles (AuNPs), resulting in color variations. The utilization of the allosteric probe, which is integrated with the aptamer sequences, enables wash-free detection of P. aeruginosa. Taking the merit of multiple signal amplification process, the suggested method showed a strong linear response to the extracted P. aeruginosa within a concentration range of 10–105 cfu/mL, with a low limit of detection for individual P. aeruginosa detection. The proposed technique has considerable clinical promise for early disease diagnosis because to its high sensitivity and wash-free simplicity.

CRISPR-Cas12a-mediated colorimetric aptasensor of netilmicin based on enzymes-assisted signal amplification and nanozyme employing a rationally truncated aptamer

Netilmicin (NET) is a typical veterinary antibiotic used in aquaculture and animal husbandry, but its excessive accumulation poses a serious threat to the ecological environment and human health. Therefore, a novel and highly sensitive Crispr-Cas12a-mediated colorimetric aptasensor was developed by combining the enzyme-assisted signal amplification strategy and magnetic NMOF-Pt nanozyme composite material (NPSM) for the visual detection of veterinary antibiotic residues in real samples. A high-affinity truncated aptamer (N-20) targeting NET was evolved from a parental aptamer sequence with the help of comprehensive recognition mechanism studies. Subsequently, N-20 was elaborately designed into the isothermal enzyme-assisted strand displacement amplification reaction. The amplified products (Act-DNAs) activated the trans-cleavage activity in the Crispr Cas12a system. The NMOF-Pt nanozyme was released from NPSM after cleaving the single chain of DNA (ssDNA). The quantity of released nanozyme was positively correlated with the amount of NET to quantitatively achieve the detection with the linear range and limits of detection (LOD) of about 0.003–30 ng/mL and 1.15 pg/mL, respectively. This method was successfully applied to detect NET in Lake water, milk and pork samples with satisfactory recoveries ranging from 89.9 % to 104.3 %. The constructed aptasensor demonstrated superior analytical performance and exhibited enormous application prospects in food supervision, environment monitoring, and medical laboratory science.

Integration of high-entropy nanozyme and hollow In2S3 nanotube heterostructures decorated with WO3 for ultrasensitive PEC aptasensing of highly toxic mycotoxin

As a highly toxic mycotoxin, patulin threatens human and animal health. Herein, a new signal amplification strategy was designed for photoelectrochemical (PEC) analysis of patulin based on nanotube-like In2S3/WO3 heterostructures with the assistance of PtRuFeCoW high-entropy alloy nanorods (HEANRs). Specifically, the prominent photoactivity of the In2S3/WO3 was investigated by UV−vis diffuse reflectance and photoluminescence spectroscopy, coupled by elaborating the photoelectron transport mechanism in detail. Simultaneously, the excellent peroxidase-like activity of the PtRuFeCoW HEANRs was certificated mainly by UV–vis absorption spectroscopy. On such foundation, the PtRuFeCoW HEANRs modified capture DNA was cast onto the In2S3/WO3 based photoanode for catalyzing 4-chloro-1-naphthol (4-CN) oxidation to form insoluble benz-4-chloro-hexadienone (4-CD) precipitate in the presence of H2O2, ultimately weakening the PEC currents. The resulting PEC sensor exhibited a wider detection range from 0.1 pg mL−1 to 500 ng mL−1 and a lower detection limit was down to 0.063 pg mL−1 (S/N = 3). The HEANRs nanozyme assisted PEC method holds great promise for ultrasensitive detection of environmental pollutants in practice.

Fluorescence Sensing of Eclampsia Biomarkers via the Immunosorbent Atom Transfer Radical Polymerization Assay.

Accurate and quantitative detection of pre-eclampsia markers is crucial in reducing pregnancy mortality rates. This study introduces a novel approach utilizing a fluorescent biosensor by the immunosorbent atom transfer radical polymerization (immuno-ATRP) assay to detect the pre-eclampsia protein marker CD81. The critical step used in this sensor is the novel signal amplification strategy of fluorescein polymerization mediated by ferritin-enhanced controlled radical polymerization, which combines with a traditional enzyme-linked immunosorbent assay (ELISA) to further reduce the detection limit of the CD81 protein concentration. The fluorescence intensity was linear versus logarithmic CD81 protein concentration from 0.1 to 10,000 pg mL-1, and the detection limit was 0.067 pg mL-1. Surprisingly, in 30% normal human serum (NHS), the sensor can also detect target protein over 0.1-10,000 pg mL-1, with 0.083 pg mL-1 for the detection limit. Moreover, the proposed biosensor is designed to be cost-effective, making it accessible, particularly in resource-limited settings where expensive detection techniques may not be available. The affordability of this method enables widespread screening and monitoring of preeclampsia, ultimately benefiting many pregnant women by improving their healthcare outcomes. In short, developing of a low-cost and susceptible direct detection method for preeclampsia protein markers, such as CD81, through the use of the immuno-ATRP assay, has significant implications for reducing pregnancy mortality. This method holds promise for early detection, precise treatment, and improved management of preeclampsia, thereby contributing to better maternal and fetal health.

A novel electrochemical aptasensor based on AgPdNPs/PEI-GO and hollow nanobox-like Pt@Ni-CoHNBs for procymidone detection

Herein, an aptasensor based on a signal amplification strategy was developed for the sensitive detection of procymidone (PCM). AgPd nanoparticles/Polenimine Graphite oxide (AgPdNPs/PEI-GO) was weaned as electrode modification material to facilitate electron transport and increase the active sites on the electrode surface. Besides, Pt@Ni-Co nanoboxes (Pt@Ni-CoHNBs) were utilized to be carriers for signaling tags, after hollowing ZIF-67 and growing Pt, the resulting Pt@Ni-CoHNBs has a tremendous amounts of folds occurred on the surface, enables it to carry a larger quantity of thionine, thus amplify the detectable electrochemical signal. In the presence of PCM, the binding of PCM to the signal probe would trigger a change in electrical signal. The aptasensor was demonstrated with excellent sensitivity and a low detection limit of 0.98 pg·mL−1, along with a wide linear range of 1 μg·mL−1 to 1 pg·mL−1. Meanwhile, the specificity, stability and reproducibility of the constructed aptasensor were proved to be satisfactory.

An ultrasensitive photoelectrochemical biosensor based on AgBiS2/CdS photoanode and multiple signal amplification strategy for the detection of dibutyl phthalate plasticizer

Herein, a novel AgBiS2/CdS heterojunction was developed for the construction of sensitive biosensor for dibutyl phthalate (DBP) detection. Compared with the pristine CdS, the AgBiS2/CdS heterojunction exhibited enhanced photocurrent because of the promoted photo-carriers transfer due to the matched energy band arrangement between AgBiS2 and CdS. Based on this AgBiS2/CdS photoanode and a signal amplification strategy integrates DNAzyme-assisted walker reaction and hybridization chain reaction, a sensitive PEC sensing platform was designed for the detection of DBP. A wide detection ranges from 1 fM to 10 nM and a low detection limit of 0.3 fM were achieved. The proposed PEC biosensor with satisfactory sensitivity, selectivity, and stability shows potential application in environmental monitoring.

A Stable and Sensitive Engineering Bacterial Sensor via Physical Biocontainment and Two-Stage Signal Amplification.

Although engineering bacterial sensors have outstanding advantages in reflecting the actual bioavailability and continuous monitoring of pollutants, the potential escape risk of engineering microorganisms and lower detection sensitivity have always been one of the biggest challenges limiting their wider application. In this study, a core-shell hydrogel bead with functionalized silica as the core and alginate-polyacrylamide as the shell have been developed not only to realize zero escape of engineered bacteria but also to maintain cell activity in harsh environments, such as extremely acidic/alkaline pH, high salt concentration, and strong pressure. Particularly, after combining the selective preconcentration toward pollutants by functionalized core and the positive feedback signal amplification of engineering bacteria, biosensors have realized two-stage signal amplification, significantly improving the detection sensitivity and reducing the detection limit. In addition, this strategy was actually applied to the detection of As(III) and As(V) coexisting in environmental samples, and the detection sensitivity was increased by 3.23 and 4.39 times compared to sensors without signal amplification strategy, respectively, and the detection limits were as low as 0.39 and 0.86 ppb, respectively.

Recent Trends and Innovations in Bead-Based Biosensors for Cancer Detection.

Demand is strong for sensitive, reliable, and cost-effective diagnostic tools for cancer detection. Accordingly, bead-based biosensors have emerged in recent years as promising diagnostic platforms based on wide-ranging cancer biomarkers owing to the versatility, high sensitivity, and flexibility to perform the multiplexing of beads. This comprehensive review highlights recent trends and innovations in the development of bead-based biosensors for cancer-biomarker detection. We introduce various types of bead-based biosensors such as optical, electrochemical, and magnetic biosensors, along with their respective advantages and limitations. Moreover, the review summarizes the latest advancements, including fabrication techniques, signal-amplification strategies, and integration with microfluidics and nanotechnology. Additionally, the challenges and future perspectives in the field of bead-based biosensors for cancer-biomarker detection are discussed. Understanding these innovations in bead-based biosensors can greatly contribute to improvements in cancer diagnostics, thereby facilitating early detection and personalized treatments.

Highly sensitive and label-free immunoassay of chloramphenicol based on poly-adenine-mediated spherical nucleic acid and hybridization chain reaction amplification

In this paper, a label-free fluorescent immunoassay for chloramphenicol was established based on a DNA-assisted signal amplification strategy. The poly-adenine mediated spherical nucleic acid was firstly employed as the substrate of antibody to construct the immunonanoprobes. Enzyme-free signal amplification techniques, hybridization chain reaction and strand displacement reaction, were utilized to develop the dual amplification strategy. The output signal was expressed through the use of G-quadruplex to achieve label free fluorescence changes. Under optimized conditions, the detection limit of this method was reduced to 0.0044 ng/L. The method exhibited a high accuracy to the detection of chloramphenicol in milk samples with a recovery rate from 94.5% to 118.1%. However, the limitations of this method probably lied in the overlong detection time and the possible interference from other compounds present in food samples. The developed label-free fluorescent immunoassay could provide an innovative view for the reliable and ultrasensitive detection of chloramphenicol, which is conducive to the food safety.

Aptamer-based protein molecule detection via cyclic reverse transcription coupling with self-priming hairpin-triggered CRISPR-Cas12a system

Protein biomarkers (e.g. thrombin) are of great significance for the biological process of the organism, and its aberrant expression is closely associated with the development of diseases. With thrombin, a serine protease that plays a crucial role in maintaining homeostasis and promoting blood clotting, as detection target, this study introduces a novel approach for sensitive and accurate measurement of protein biomarker expression by utilization of cyclic reverse transcription (CRT) in combination with the self-priming hairpin-triggered CRISPR-Cas12a system. In this method, an elegantly designed sensing probe is utilized to specifically bind with the thrombin protein and convert the protein signals to nucleic acids signals, following by the CRT and CRISPR-Cas12a system-based signal amplification strategy. Taking the merit of the two-stage amplification, this assay has the capability to detect thrombin at the fM level. In addition, due to the aptamer sequence’s strong selectivity to thrombin protein and the dual-check process in the signal amplification process (first in the CRT and second in the CRISPR system), the proposed test demonstrates exceptional specificity in detecting thrombin. By re-designing the sensing probe, the established method could be extended to various protein biomarker detection. Ultimately, this assay has successfully enabled the accurate evaluation of biomarker levels in constructed clinical samples, showing significant potential for application in the realm of clinical molecular diagnosis.