Triggered Hybridization Chain Reaction Research Articles

A triple-signal strategy separated aptasensor for sensitive determination of aflatoxin B1 in peanut based on electrochemical and fluorescent dual-mode

It is necessary to develop accurate and sensitive AFB1 detection method due to its high toxicity of pathogenicity and carcinogenicity. The results of traditional single-mode AFB1 detection methods are easily affected by the test environment and human operation. Herein, an electrochemical and fluorescent dual-mode separated aptasensor was developed for detecting AFB1 with triple-signal readout. The AFB1 aptasensor was constructed by combining the Fc-labeled AFB1 aptamer with its complementary DNA (cDNA). Subsequently, the methylene blue (MB) was incorporated into dsDNA as another signal tag. In the presence of AFB1, the Fc tag approached to electrode surface, both of the cDNA and MB molecule released due to the interaction between AFB1 and its aptamer, resulting in “signal-on” and “signal-off” dual-signal strategy. There was a good linear relationship between the SWV electrochemical response signal and the logarithm of AFB1 concentration at 0.01–200 ng/mL (LOD 2.5 pg/mL, S/N = 3). Meanwhile, the released cDNA was designed to trigger hybridization chain reaction (HCR), and the HCR product was served as template for the formation of fluorescent copper nanoparticles (CuNPs). There was a good linear relationship between the fluorescent intensity of CuNPs and the logarithm of AFB1 concentration at 0.001–200 ng/mL (LOD 0.28 pg/mL, S/N = 3). The separated fluorescent method showed broadened linear range and lower LOD compared with SWV method, both up to 10-folds, which improved the detection sensitivity. The constructed dual-mode AFB1 aptasensor showed satisfactory selectivity, stability and reproducibility, and the good recoveries of real peanut samples indicated its practical application in food detection.

Split aptamer-based sandwich-type ratiometric biosensor for dual-modal photoelectrochemical and electrochemical detection of 17β-estradiol

BackgroundAs one of the most potent environmental estrogens, 17β-estradiol (E2), which can be enriched into organisms through the food chain and cause harmful biological effects in humans, has been frequently detected in the water environment of the world. High performance liquid chromatography (HPLC) and gas chromatograohy-mass spectrometry (GC/MS) have been widely used for quantification of E2. Despite excellent accuracy, tedious pretreatment and expensive instruments result in their limited application. It is clear that there is an urgent need to establish simple, sensitive and accurate methods for the determination of E2. ResultsA split aptamer-based sandwich-type ratiometric biosensor based on split aptamer was developed by coupling photoelectrochemical and electrochemical assays for E2 detection. For analysis, the two fragments of split aptamer recognized E2 by forming sandwich structure, which triggered hybridization chain reaction (HCR) to produce double-stranded DNA (dsDNA) with CdTe quantum dots (QDs) labeled hairpin DNA. The resultant dsDNA can further absorb methylene blue (MB) to sensitize CdTe QDs for an enlarged photocurrent (IPEC) and output a redox current of IMB, and both of them acted as response signals for detection; [Fe(CN)6]3-/4- probe produced redox current of I[Fe(CN)6]3-/4- as reference signal. Using IMB/I[Fe(CN)6]3-/4- and IPEC/I[Fe(CN)6]3-/4- as yardsticks, the developed split aptamer-based sandwich-type ratiometric biosensor provides two linear ranges of 0.1–5000 pg mL−1 for IMB/I[Fe(CN)6]3-/4- and 0.1−10000 pg mL−1 for IPEC/I[Fe(CN)6]3-/4- with detection limits of 0.06 pg mL−1 and 0.02 pg mL−1, respectively. SignificanceThese results of the biosensor are benefiting from the coupling of photoelectrochemical (PEC) and electrochemical (EC) assays as well as the unique cooperative recognition mechanism of split aptamer. This method not only enabled the biosensor to be successfully applied to the determination of E2 in lake water, but also broadens the prospects for the realization of sensitive and accurate detection of E2.

DNAzyme-driven tripedal DNA walker triggered hybridization chain reaction for label-free electrochemical detection of Alzheimer’s tau protein

The tau protein is considered to be a reliable molecular biomarker of Alzheimer's disease (AD). Developing sensitive and reliable techniques for tau protein detection is a vital task. In this work, we designed a label-free electrochemical aptasensor for tau protein quantification based on Mg2+-DNAzyme (MNAzyme)-driven tripedal DNA walker. With the assistance of MNAzyme, free-running tripedal DNA walkers could rapidly and continually scissor DNA molecular beacon (MBs), and the fragment as a primer initiated the hybridization chain reaction (HCR) process generating a huge amount of long DNA strand containing many adenines. Following that, the strand with long-repeated adenines (HCR product) may absorb numerous AgNPs on the surface of electrode for electrochemical stripping analysis. Benefiting from MNAzyme-tripedal DNA walker and HCR triple signal amplification, we achieved a superior detection limit of 0.43 fM as well as a broad linear range of 1 fM to 0.1 nM. After being applied to tau protein detection in human serum samples, this novel electrochemical aptasensor demonstrated a high degree of anti-interference, and reproducibility, indicating its great potential for bioanalysis in complex biological environments.

Two colors, one-step, self-drive fluorescent strategy for chloramphenicol detection base on DNAzyme cleavage triggered hybridization chain reaction

A two colors, one-step, self-drive fluorescent strategy was developed for chloramphenicol (CAP) detection based on cyclic cleavage of molecular beacon (MB) by pincer DNA sequences. CAP can bind with its aptamer and active the enzyme-strand (E-DNA). Then the E-DNA can circularly cleave the MB on the both side of pincer DNA sequences. The cleaved fragments can self-assembly to form a long duplex and cause the great recovery of the two colors fluorescent signal. The limit of detection was as low as 0.7 pM. Importantly, the whole detection process is very simple with only one-step operation. Moreover, the two colors fluorescent signals can greatly enhance the accuracy of the result. It was also successfully used to detect CAP in actual samples.

Programmable Analytical Feature of Ratiometric Electrochemical Biosensor by Alternating the Binding Site of Ferrocene to DNA Duplex for the Detection of Aflatoxin B1†

Comprehensive SummaryRatiometric biosensor is among the promising strategies for the advanced analysis; its applicability in multiple scenarios, however, is seriously limited for the fixed analytical feature. Here, we reported ratiometric electrochemical biosensors with programmable analytical feature by alternating the binding site of ferrocene (Fc) to DNA duplex for the detection of aflatoxin B1 (AFB1). DNA duplex was synthesized with AFB1 aptamer and its complementary DNA (cDNA) with Fc molecular tagged on aptamer or cDNA. For analysis, aptamer‐AFB1 complex stripped from the electrode, and free cDNA triggered hybridization chain reaction to produce double‐stranded DNA for methylene blue (MB) absorption. In this way, redox currents of Fc (IFc) and absorbed MB (IMB) at the electrode were collected to produce the ratiometric signal of IMB/IFc as a yardstick for AFB1. When Fc was tagged on aptamer, IFc decreased with higher concentration of AFB1 (cAFB1), the resultant biosensor allowed detection with an On/Off (IMB/IFc) mode; when Fc was tagged on cDNA, IFc kept constant as cAFB1 varying, offering an On/Constant (IMB/IFc) mode for the detection. Consequently, biosensor with On/Off mode showed a linear range of 0.1—100 pg/mL with a limit of detection (LOD) of 0.0650 pg/mL, while biosensor with On/Constant mode offered a wider linear rang of 1—10000 pg/mL but a relatively poor LOD of 0.330 pg/mL. Our work revealed the vital importance of binding site of probes to DNA, and this principle can endow ratiometric biosensor with programmable analytical feature (e.g., linear range, LOD), allowing detections in various scenarios.

Ultrasensitive electrochemiluminescence biosensors based on programmable aptazyme-induced hybridization chain reaction for detecting adenosine triphosphate and quinine

Biosensors with programmability and universal applicability that simplify the detection of biomolecules are highly valuable. Herein, a programmable-component electrochemiluminescence biosensor based on an aptazyme-induced hybridization chain reaction and DNA-templated silver nanoclusters has been developed. An aptazyme-induced hybridization chain reaction system was designed, wherein aptazymes composed of a split aptamer and RNA-cleaving DNAzyme could recognize the target and regulate the activity of DNAzyme. Aptazymes activated by the target cyclically cleaved the substrate to produce short-ssDNA as converted signals, which were captured by engineering tetrahedral-DNA to trigger hybridization chain reaction, hence forming long dsDNA with nicks on the surface electrode. These dsDNA molecules were pre-programmed with cytosine (C)-rich sequences at the nick site, and silver nanoclusters could be synthesized in situ, generating an amplified electrochemiluminescence signal. Thus, the constructed multilevel-amplified adenosine triphosphate (ATP) electrochemiluminescence aptasensor showed good sensitivity, selectivity, and reconfiguration with a linear range from 0.1 pM to 10 nM and a limit of detection of 38.2 fM. Subsequently, an electrochemiluminescence biosensor for the detection of quinine was constructed using the same design, by replacing the ATP aptamer of aptazymes with the quinine aptamer. The quinine electrochemiluminescence biosensor was also significantly sensitive with a linear range from 10 pM to 1 μM and a limit of detection of 3.7 pM, indicating flexibility of the proposed strategy, which can be extended to fabricate other aptasensors.

Ultrasensitive Detection of Ochratoxin A With a Zeolite Imidazolate Frameworks Composite–Based Electrochemical Aptasensor

Ochratoxin A (OTA) is a harmful mycotoxin, which is mainly secreted by Penicillium and Aspergillus species. In this work, an electrochemical aptasensor is presented for OTA detection based on Au nanoparticles (AuNPs) modified zeolite imidazolate frameworks (ZIFs) ZIF-8 platform and duplex-specific nuclease (DSN) triggered hybridization chain reaction (HCR) signal amplification. G-quadruplex-hemin assembled HCR nanowire acted as a nicotinamide adenine dinucleotide (NADH) oxidase and an HRP-mimicking DNAzyme. Besides, thionine (Thi) was enriched as a redox probe for signal amplification in this pseudobienzyme electrocatalytic system. Under the optimal conditions, the analytical response ranged from 1 to 107 fg ml−1 with a detection limit of 0.247 fg ml−1. Furthermore, the aptasensor was proven to be applied in real wheat samples with a recovery between 96.8 and 104.2%, illustrating the potential prospects in practical detection.

A functionalized magnetic nanoparticle regulated CRISPR-Cas12a sensor for the ultrasensitive detection of alpha-fetoprotein.

Alpha-fetoprotein (AFP) is an important clinical tumor marker of hepatoblastoma, and the concentration of AFP in serum is closely related to the staging of hepatoblastoma. We report a magnetic bead separation platform based on a switching aptamer triggered hybridization chain reaction (SAT-HCR) and the CRISPR-Cas12a sensor for the in vitro detection of AFP. AFP aptamer, as an easily regulated nucleic acid strand, is responsible for binding to AFP into nucleic acid detection, while HCR-CRISPR-Cas12a, regulated by functionalized magnetic nanoparticles, is responsible for highly specific nucleic acid signal amplification. Under the optimal conditions, the fluorescence intensity was proportional to the concentration of AFP in the range of 0.5-104 ng mL-1 and the limit of detection was 0.170 ng mL-1. In addition, we have successfully applied this biosensor to detect AFP in clinical samples from patients with hepatoblastoma, with greater sensitivity relative to ELISA. Our proposed method showed great potential application in clinical diagnosis and pharmaceutical-related fields with the properties of high sensitivity, low cost and high selectivity.

Two Colors, One-Step, Self-Drive Fluorescent Strategy for Chloramphenicol Detection Base on Dnazyme Cleavage Triggered Hybridization Chain Reaction

Two Colors, One-Step, Self-Drive Fluorescent Strategy for Chloramphenicol Detection Base on Dnazyme Cleavage Triggered Hybridization Chain Reaction

Mechanically Triggered Hybridization Chain Reaction.

Cells transmit piconewton forces to receptors to mediate processes such as migration and immune recognition. A major challenge in quantifying such forces is the sparsity of cell mechanical events. Accordingly, molecular tension is typically quantified with high resolution fluorescence microscopy, which hinders widespread adoption and application. Here, we report a mechanically triggered hybridization chain reaction (mechano-HCR) that allows chemical amplification of mechanical events. The amplification is triggered when a cell receptor mechanically denatures a duplex revealing a cryptic initiator to activate the HCR reaction in situ. Importantly, mechano-HCR enables direct readout of pN forces using a plate reader. We leverage this capability and measured mechano-IC50 for aspirin, Y-27632, and eptifibatide. Given that cell mechanical phenotypes are of clinical importance, mechano-HCR may offer a convenient route for drug discovery, personalized medicine, and disease diagnosis.

Mechanically Triggered Hybridization Chain Reaction

AbstractCells transmit piconewton forces to receptors to mediate processes such as migration and immune recognition. A major challenge in quantifying such forces is the sparsity of cell mechanical events. Accordingly, molecular tension is typically quantified with high resolution fluorescence microscopy, which hinders widespread adoption and application. Here, we report a mechanically triggered hybridization chain reaction (mechano‐HCR) that allows chemical amplification of mechanical events. The amplification is triggered when a cell receptor mechanically denatures a duplex revealing a cryptic initiator to activate the HCR reaction in situ. Importantly, mechano‐HCR enables direct readout of pN forces using a plate reader. We leverage this capability and measured mechano‐IC50 for aspirin, Y‐27632, and eptifibatide. Given that cell mechanical phenotypes are of clinical importance, mechano‐HCR may offer a convenient route for drug discovery, personalized medicine, and disease diagnosis.

Highly sensitive homogeneous electrochemiluminescence biosensor for microRNA-21 based on cascaded signal amplification of target-induced hybridization chain reaction and magnetic assisted enrichment

MicroRNA-21(miRNA-21) can act as a novel biomarker in early stage of acute kidney injury (AKI). The development of simple but sensitive miRNA-21 detection technology is helpful for prevention and treatment of AKI. In this work, based on the cascaded signal amplifications of the target-induced hybridization chain reaction (HCR) and magnetic assisted enrichment, a highly sensitive homogeneous electrochemiluminescence (ECL) biosensor for miRNA-21 detection had been developed. Firstly, capture probe H1 (CPH1) had been modified on magnetic beads (MBs) which could capture target (miRNA-21) and trigger HCR amplification to produce long strand dsDNA in the homogeneous solution. Ru(phen)32+ could be embedded into the grooves of dsDNA and played the role of luminous tag, which could be separated by magnetic easily to realize the enrichment. Then the enriched products had been dissolved in the buffer solution and the ECL signal could be detected easily. The ECL intensity had a linear correlation with the logarithm of miRNA-21 concentration in the range of 1.0 fM-1.0 nM, the detection limit was 0.14 fM. The method developed in this work showed well sensitivity, specificity, which had been applied to detect miRNA-21 in urine of AKI patients with satisfied results.

A competitive colorimetric aptasensor transduced by hybridization chain reaction-facilitated catalysis of AuNPs nanozyme for highly sensitive detection of saxitoxin

Saxitoxin (STX) is a small molecule toxin (Mw. ca. 299 g/mol) with high acute toxicity, and it has urgent need of facile analytical methods. Herein, a competitive colorimetric aptasensor was developed for highly sensitive detection of STX. An anti-STX aptamer was hybridized with a complementary strand on the magnetic beads and was competitively bound by STX. The supernatant containing the aptamer binding to STX was obtained by magnetic separation, which could trigger hybridization chain reaction (HCR) to generate rigid double stranded DNAs (dsDNAs) with sticky end and variable length. These HCR-dsDNAs were found to be able to facilitate significant enhancement on the peroxidase-like catalytic capability of AuNPs nanozyme towards 3,3,5,5-tetramethylbenzidine (TMB). The concentration of STX was responded in a “turn on” mode, based on the amplified colorimetric transduction thereof. The aptasensor realized high sensitivity, with a limit of detection (LOD) as low as 42.46 pM. Moreover, a wide linear detection range of 78.13–2500 pM, good selectivity, as well as good recovery rates of 106.2–113.5% when analyzing STX in real shellfish samples were obtained. This strategy could be referred to develop robust aptasensors for simple and highly sensitive detection of other small molecules and toxins.

Proximity hybridization triggered hybridization chain reaction for label-free electrochemical homogeneous aptasensors

A label-free homogeneous electrochemical aptasensor was developed for detection of thrombin based on proximity hybridization triggered hybridization chain reaction induced G-quadruplex formation. Thrombin promoted the formation of a complex via the proximity hybridization of the aptamer DNA strands, which unfolded the molecular beacon, the stem part of molecular beacon as a primer to initiate the hybridization chain reaction process. Thus, with the electrochemical indicator hemin selectively intercalated into the multiple G-quadruplexes, a significant electrochemical signal drop is observed, which is dependent on the concentration of the target thrombin. Thus, using this“signal-off” mode, label-free homogeneous electrochemical strategy for sensitive thrombin assay with a detection limit of 44 fM is realized. Furthermore, this method also exhibits additional advantages of simplicity and low cost, since both expensive labeling and sophisticated probe immobilization processes are avoided. Its high sensitivity, acceptable accuracy, and satisfactory versatility of analytes led to various applications in bioanalysis.

Non-invasive diagnosis of bladder cancer by detecting telomerase activity in human urine using hybridization chain reaction and dynamic light scattering

Cystoscopy and histology are the gold standards for detection of bladder cancer. However, these methods are highly subjective, expensive, and invasive. We have developed a non-invasive method for the diagnosis of bladder cancer by detecting telomerase activity in human urine. Telomerase substrate (TS) primer is elongated with repeating sequences of (TTAGGG)n in the presence of telomerase. The elongated primer can trigger hybridization chain reaction between two hairpins H1 and H2, result in the aggregation of AuNPs due to the hybridization between the tail sequence on H1 (or H2) and DNA-AuNPs probe, and accompany with the increase of hydrodynamic diameter of AuNPs, which can be measured with dynamic light scattering (DLS). The biosensor displayed a detection limit of 4 MCF-7 cells (a signal-to-noise ratio of 3) and a dynamic range of 10–1000 cells. Moreover, only urine specimens from bladder cancer patients induced a significant change in the average hydrodynamic diameter, indicating its specificity for the non-invasive diagnosis of bladder cancer.

Dandelion-like CuO microspheres decorated with Au nanoparticle modified biosensor for Hg2+ detection using a T-Hg2+-T triggered hybridization chain reaction amplification strategy

We fabricate a novel electrochemical biosensor based on the specific thymine-Hg2+-thymine (T-Hg2+-T) base pair for the highly sensitive detection of mercury ions (Hg2+) and utilize toluidine blue (TB) as a redox indicator that is combined with a hybridization chain reaction (HCR) for signal amplification. The dandelion-like CuO (D-CuO) microspheres that were assembled using Au nanoparticles were first introduced as support materials, which produced more active sites for the thiolated probe (P1) combination. Then, the presence of Hg2+ induced P1 to hybridize with the other oligonucleotide (P2) through Hg2+-mediated T-Hg2+-T complexes. In addition, the partial sequence of P2 acted as an initiator sequence, which led the two hairpin DNA (H1 and H2) strands to collectively form the extended double-strand DNA through the HCR process on the electrode surface. TB was employed to interact with the double strands and produce an efficient electrochemical signal. The proposed strategy combined the amplification of the HCR and the inherent redox activity of TB and utilized D-CuO/Au composites, which exhibited high sensitivity for Hg2+ determination. Under the optimum conditions, the proposed biosensor showed a prominent response for Hg2+, including a linear range from 1 pM to 100 nM and a detection limit of 0.2 pM (S/N = 3). Moreover, the new biosensor proved its potential application for trace Hg2+ determination in environmental water samples.

Label-free and amplified electrogenerated chemiluminescence biosensing for the detection of thymine DNA glycosylase activity using DNA-functionalized gold nanoparticles triggered hybridization chain reaction

Effective detection of thymine DNA glycosylase (TDG) activity is extremely crucial and urgent for epigenetic research. Herein, a novel label-free electrogenerated chemiluminescence (ECL) biosensing method was developed for the detection of TDG activity using DNA-functionalized gold nanoparticles (DNA-AuNPs) triggered hybridization chain reaction (HCR). In this assay, the thiol modified hairpin probe DNA (hp-DNA) with 5′ overhangs and one mismatched base pair of guanines: thymine (G: T) in the stem part was boned onto gold electrode. TDG specifically removed T base of the G: T mismatch to produce apyrimidinic (AP) sites through the N-glycosidic bond hydrolysis. The AP site was then cleaved by the catalysis of Endonuclease IV (EnIV) to generate dsDNA containing a free 3’ end in the long sequence, which serves as a complementary sequence to hybridize with the specific sequence (ssDNA1) of DNA-AuNPs. Then, the functionalized DNA-AuNPs with initiator strands (ssDNA2) could trigger HCR to form nicked double helices DNA polymer which can embed numerous ECL indicator, Ru(phen)32+, resulting in significantly increased ECL signal. The proposed strategy combined the amplification function of DNA-AuNPs triggered HCR and the inherent high sensitivity of the ECL technique, a detection limit of 1.1 × 10−5 U/μL (0.0028 ng/mL) for TDG determination was obtained. In addition, this method was successfully applied to evaluate TDG activity in cancer cell, which provides great possibility for TDG activity assay in related clinical diagnostics.

Multiplexed chemiluminescence imaging assay of protein biomarkers using DNA microarray with proximity binding-induced hybridization chain reaction amplification

A multiplexed chemiluminescence (CL) imaging assay was designed for sensitive screen detection of a panel of protein biomarkers by integrating DNA microarray with proximity binding-induced hybridization chain reaction (HCR) amplification. The DNA microarray was manufactured by printing hairpin DNAs for different analytes detection in an array format onto the sensing cells arranged on an aldehyde-functionalized glass chip. The existence of target proteins induced the formation of sandwich immunocomplexes along with the hybridization of DNA strands labeled on the pair of antibodies via proximity effect, which subsequently unfolded their corresponding spotting hairpin DNAs to expose prelocked primer and trigger HCR assembly. Through the streptavidin-biotin conjugation, numerous horseradish peroxidase (HRP) was coupled on the HCR assemblies to catalyze H2O2-luminol reaction and produce amplified CL signals for sensitive CL imaging assay of protein targets. As a proof of concept, the DNA microarray was used to CL imaging assay of carcinoembryonic antigen, α-fetoprotein and carcinoma antigen 125 in ranges of 0.05–500 ng mL−1, 0.06–600 ng mL−1 and 0.02–200 U mL−1, respectively. The CL microarray imaging assay could also be extended to screen detect other biomarker groups by printing different hairpin DNAs on the microarray and using corresponding antibody-DNA pairs. The proposed CL microarray imaging assay showed advantages of small sample consumption, good cost effectiveness, and multiplex detection performance, exhibiting good applicability in clinical diagnosis.

High-Sensitive Electrochemiluminescence C-Peptide Biosensor via the Double Quenching of Dopamine to the Novel Ru(II)-Organic Complex with Dual Intramolecular Self-Catalysis.

Here, a novel Ru(II)-organic complex (Ru-PEI-ABEI) with high electrochemiluminescence (ECL) efficiency was proposed to construct a sensitive quenching-typed ECL biosensor for C-peptide (C-P) measurement based on the double quenching effect of dopamine (DA). The high ECL efficiency of Ru-PEI-ABEI was originated from the dual intramolecular self-catalysis including intramolecular coreaction between polyethylenimine (PEI) and Ru(bpy)2(mcbpy)2+, and intramolecular ECL resonance energy transfer (ECL-RET) from N-(aminobutyl)-N-(ethylisoluminol) (ABEI) to Ru(bpy)2(mcbpy)2+, which would generate a strong initial ECL signal. Through sandwiched immunoreaction and 3D DNA walking machine, a certain amount of target C-P was converted to a large amount of intermediate DNA that could further trigger hybridization chain reaction (HCR) to introduce into massive DA which not only could quench the ECL of Ru(bpy)2(mcbpy)2+, but also quench the ECL of ABEI. Thus, the double quenching effect of DA would effectively quench the ECL of Ru-PEI-ABEI, leading to an obviously decreased final ECL signal. Thus, a sensitive quenching-typed ECL biosensor was constructed for C-P detection with a linear range from 50 fg mL-1 to 16 ng mL-1 and an estimated detection limit of 16.7 fg mL-1. The dual intramolecular self-catalyzed strategy and the double quenching strategy based on one quencher to the same luminous reagent proposed in this work would provide new thought in both ECL signal enhancement and quenching efficiency improvement.

CdS:Mn quantum dot-functionalized g-C3N4 nanohybrids as signal-generation tags for photoelectrochemical immunoassay of prostate specific antigen coupling DNAzyme concatamer with enzymatic biocatalytic precipitation

A new photoelectrochemical (PEC) immunosensor based on Mn-doped CdS quantum dots (CdS:Mn QDs) on g-C3N4 nanosheets was developed for the sensitive detection of prostate specific antibody (PSA) in biological fluids. The signal derived from the as-synthesized Cd:Mn QDs-functionalized g-C3N4 nanohybrids via a hydrothermal method and was amplified through DNAzyme concatamers on gold nanoparticles accompanying enzymatic biocatalytic precipitation. Experimental results by UV–vis absorption spectra and photoluminescence revealed that CdS:Mn QDs/g-C3N4 nanohybrids exhibited higher photocurrent than those of CdS:Mn QDs and g-C3N4 alone. Upon addition of target PSA, a sandwich-type immunoreaction was carried out between capture antibodies and the labeled detection antibodies. Accompanying introduction of gold nanoparticles, the labeled initiator strands on the AuNPs triggered hybridization chain reaction and the formation of DNAzyme concatamers in the presence of hemin. The formed DNAzyme catalyzed 4-chloro-1-naphthol (4-CN) to produce an insoluble/insulating precipitate on the Mn:CdS QDs/g-C3N4, and blocked the light harvesting of Mn:CdS QDs/g-C3N4, thus resulting in the decreasing photocurrent. Under optimal conditions, the immunosensor exhibited good photocurrent responses for determination of target PSA, and allowed detection of PSA at a concentration as low as 3.8pgmL−1. The specificity, reproducibility and precision of this system were acceptable. Significantly, this methodology was further evaluated for analyzing human serum samples, giving well-matched results with referenced PSA enzyme-linked immunosorbent assay (ELISA) method.