Triple Signal Amplification Strategy Research Articles

Ultrasensitive electrochemical biosensor based on triple signal amplification strategy for detection of serum cf-miR-181b: A possible biomarker related to osteosarcoma’s early diagnosis and chemotherapy monitoring

Osteosarcoma is one of the most common primary malignant bone tumors, however, the current diagnostic methods based on tissue biopsy suffers from invasive operation, high heterogeneity and non-early diagnosis. Herein, we proposed a new liquid biopsy method using triple signal amplification-based electrochemical biosensor (E-sensor) in detection of serum cf-miR-181b closely related to osteosarcoma. The E-sensor adopted an enzyme-linked DNA magnetic microbead (MMB), in which the miR-181b-triggered ligase chain reaction products were immobilized onto the surface of MMB followed by the attraction of horseradish peroxidase, to produce a catalytic current on the electrode surface. The E-sensor could detect miR-181b as low as 6.70 aM with a single-base resolution. Finally, the E-sensor was applied to detect serum cf-miR-181b in different individuals, which exhibited excellent performance in discriminating serum miR-181b between control group (healthy people/non-osteosarcoma patients) and osteosarcoma patients. Also, this assay could be used to distinguish the osteosarcoma patients before and after effective chemotherapy. In parallel, all results were in good agreement with that obtained by qRT-PCR. Therefore, the proposed E-sensor, due to its low cost, easy miniaturization and generalization, holds a huge application potential in the early auxiliary diagnosis and therapeutic monitoring of osteosarcoma and is of great value in clinical translation.

Triple signal amplification strategy for ultrasensitive in situ imaging of intracellular telomerase RNA

Abnormal upregulation of telomerase RNA (TR) is a hallmark event at various stages of tumor progression, providing a universal marker for early diagnosis of cancer. Here, we have developed a triple signal amplification strategy for in situ visualization of TR in living cells, which sequentially incorporated the target-initiated strand displacement circuit, multidirectional rolling circle amplification (RCA), and Mg2+ DNAzyme-mediated amplification. All oligonucleotide probes and cofactors were transfected into cells in one go, and then escaped from lysosomes successfully. Owing to the specific base pairing, the amplification cascades could only be triggered by TR and performed as programmed, resulting in a satisfactory signal-to-background ratio. Especially, the netlike DNA structure generated by RCA encapsulated high concentrations of DNAzyme and substrates (FQS) in a local region, thereby improving the reaction efficiency and kinetics of the third amplification cycle. Under optimal conditions, the proposed method exhibited ultrasensitive detection of TR mimic with a detection limit at pM level. Most importantly, after transfection with the proposed sensing platform, tumor cells can be easily distinguished from normal cells based on TR abundance-related fluorescence signal, providing a new insight into initial cancer screening.

Ultrasensitive detection of CA125 based on a triple signal amplification strategy with a huge number of loaded probes via exonuclease cyclic cleavage, rolling cyclic amplification and strand self-growth.

A novel electrochemiluminescence (ECL) aptamer biosensor with high sensitivity and selectivity for the detection of tumor biomarker carbohydrate antigen 125 (CA125) was constructed, and a strategy of triple amplification of signals was proposed using an exonuclease cyclic cleavage aptamer, combined with rolling ring amplification technologies, generating multi-branched dendritic double-stranded DNA to load a large number of probes through "strand self-growth". The double-stranded DNA, which is abbreviated as CP/CA dsDNA, formed by hybridizing the single strand of capture DNA (CP DNA) with the single strand DNA of the CA125 aptamer (CA Apt) was modified on Fe3O4@Au. When CA125 was added, CP/CA dsDNA was unwound, and CA125 specifically combined with CA Apt to form a protein-aptamer complex, leaving only CP DNA on the surface of Fe3O4@Au. RecJf exonuclease cleaved the aptamer in the protein-aptamer complex and released CA125, which recombined with other CA125 aptamers, to form a cycle that produces more CP DNA on Fe3O4@Au. Three ssDNA (H1, H2, and H3) were introduced and hybridized with CP DNA to form a dsDNA with a "+" configuration structure. Then phi29 DNA polymerase, T4 DNA ligase, deoxy-ribonucleoside triphosphate (dNTP) and padlock probes were added to form a large number of complementary strands of padlock probes (CS padlock probes) by rolling cyclic amplification. CS padlock probes were linked to the "+" type dsDNA; then ssDNA H4 was added and hybridized with the CS padlock probe to form multi-branched dendritic dsDNA. A large number of tris(2,2'-bipyridyl)ruthenium(II) probes were embedded in the double strands, resulting in an extremely strong ECL signal in the presence of the co-reactant tri-n-propylamine (TPA). There is a linear relationship between the ECL signals and CA125 concentrations in the range of 1.0 × 10-15-1.0 × 10-8 mg mL-1, and the detection limit was 2.38 × 10-16 mg mL-1. It has been used for the determination of CA125 in serum samples.

Electrochemical analysis of microRNAs with hybridization chain reaction-based triple signal amplification

Selective and sensitive detection of trace microRNA is important for early diagnosis of diseases due to its expression level related to diseases. Herein, a triple signal amplification strategy is developed for trace microRNA-21 (miRNA-21) detection by combining with target-triggered cyclic strand displacement reaction (TCSDR), hybridization chain reaction (HCR) and enzyme catalytic amplification. Four DNA hairpins (H1, H2, H3, H4) are employed to form an ultralong double-strand DNA (dsDNA) structure, which is initiated by target miRNA-21. As H3 and H4 are labeled with horseradish peroxidase (HRP), numerous HRPs are loaded on the long dsDNA, producing significantly enhanced electrocatalytic signals in the hydrogen peroxide (H2O2) and 3,3′,5,5′-tetramethylbenzidine (TMB) reaction strategy. Compared with single signal amplification, the triple signal amplification strategy shows higher electrochemical response, wider dynamic range and lower detection limit for miRNA-21 detection with excellent selectivity, reproducibility and stability. Taking advantage of the triple signal amplification strategy, the proposed electrochemical biosensor can detect miRNA-21 in 10 HeLa cell lysates, suggesting that it is a promising method for fruitful assay in clinical diagnosis.

A novel “signal on-off-super on” sandwich-type aptamer sensor of CRISPR-Cas12a coupled voltage enrichment assay for VEGF detection

Vascular endothelial growth factor (VEGF) plays an important role in atherosclerosis, and the detection of VEGF is critical for the prevention, monitoring, and diagnosis of cardiovascular diseases. Here, a novel “signal on-off-super on” sandwich-type aptamer sensor with a triple signal amplification strategy was developed for the first time. Based on the capture aptamer was labeled with methylene blue (MB) on the internal bases, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a-coupled voltage enrichment was used to amplify the electrochemical signal. To improve the analytical performance of the aptamer sensor, gold nanoparticles@Ti3C2Tx-Mxene (AuNPs@Ti3C2Tx-Mxene) were synthesized through the electrodeposition of AuNPs on the Ti3C2Tx-Mxene surface, providing active sites for the immobilization of the aptamer and amplifying the electrochemical signals. The excellent trans-cleavage activity of the CRISPR-Cas12a system was harnessed to cleave signal probes. The cleaved signal probes were enriched using an electrochemical signal instead of complicated target amplification steps before detection. Hence, we report a simplified detection process for amplifying electrochemical signals. Under optimal conditions, the aptamer sensor exhibited high sensitivity, acceptable stability, and reproducibility with a wide linear range from 1 pM to 10 μM (R2 = 0.9917) and an ultralow detection limit of 0.33 pM (S/N = 3). Therefore, we propose a novel strategy of CRISPR-Cas12a-based protein detection that opens a new window for the diagnostic applications of various biomarkers.

Picomolar thrombin detection by orchestration of triple signal amplification strategy with hierarchically porous Ti3C2Tx MXene electrode material-catalytic hairpin assembly reaction-metallic nanoprobes

Signal amplification strategies are essential to boost the sensitivity of detecting targeted ions or molecules with important biological functions, while few studies take advantage of signal amplification strategies more than two. As a “proof-of-concept” demonstration, we present the ultrasensitive electrochemical aptasensor for picomolar thrombin detection by synchronous coordination of triple signal amplification strategy. The porous MXene framework (PMXF) with secondary pores is constructed as carrier to increase electrons transfer channels, and thionine (as redox indicator) labelled Au nanorod (AuNR) and hollow Cu–Pt alloy (HCuPtA) are synthesized as the electrical signal amplifiers to enhance the response signals. In the presence of picomolar-level thrombin, catalytic hairpin assembly reactions of DNA are triggered to bridge thionine labelled AuNR or HCuPtA nanoprobes on the PMXF with controllablly scondary pore structures. Under the optimal conditionals, the sandwich-typed aptasensor based on PMXF-5/AuNR shows a more low limit of detection (LOD) of 0.67 pM with a linear range from 2 pM to 10 nM, while PMXF-5/HcuPtA exhibits a more wide linear range from 50 pM to 50 nM with a LOD of 16.67 pM for thormbin. This sensing platform can be customized to analyze other biological or environmental substances at an ultrahigh level by rationally designing DNA sequences of target-binding aptamer.

A highly sensitive electrochemical cytosensor based on a triple signal amplification strategy using both nanozyme and DNAzyme.

The development of a sensitive cytosensor is beneficial for the early diagnosis and treatment of cancer. Herein, highly sensitive cytosensing was achieved by applying triple signal amplification strategies with Fe3O4@Au nanozymes and DNAzyme hybrids as electrochemical nanoprobes and toluidine blue (Tb) as the electron transfer medium. The Fe3O4@Au nanocomposites not only acted as nanozymes with excellent catalytic performance towards H2O2 reduction but also served as promising scaffolds to carry massive electroactive substances and DNA probes. The dual-functional DNA probes were designed with the sequence of hemin/G-quadruplex to serve as the DNAzyme and the sequence of aptamer to recognize cancer cells. Furthermore, Tb was also conjugated to the surface of the Fe3O4@Au nanohybrids, working as the electron transport medium to magnify the electrochemical response. With the above design, the Fe3O4@Au nanozymes and hemin/G-quadruplex DNAzyme efficiently co-catalyzed the reduction of H2O2 to accelerate the electron transfer of Tb, which realized triple signal amplification and finally improved the performance of the electrochemical cytosensor. The proposed cytosensor achieved a sensitive detection of HepG2 cells with a low detection limit of 20 cells mL-1, and could be potentially used as an effective analysis tool in early cancer diagnosis in the future.

Rapid heavy metal sensing platform: A case of triple signal amplification strategy for the sensitive detection of serum copper

Heavy metals are considered as hazardous substances to human because of their toxicity, persistence and bioaccumulation, and the level in serum is an important factor to evaluate the caused health risk, which depends on efficient and sensitive analytical methods. Here, a triple signal-amplified electrochemical sensing platform based on metal-dependent DNAzymes was fabricated for sensitive determination of heavy metals in serum (copper as a model target). Under the optimized conditions, the proposed method showed good sensitivity (limit of detection, 0.33 fM for Cu2+) with excellent selectivity and stability, which is ascribed to: (i) tetrahedral DNA nanostructures (TDNs) that was used as a promising scaffold to adjust the selective transformation between heterogeneous and homogeneous reactions, preventing the nonspecific binding of electrodes surface and DNA probes; (ii) the magnetic beads (MBs) used which led to signal amplification and decreased background owing to its excellent properties of extracting equivalent targets from the complex samples; (iii) two signal amplification strategy of catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR). In addition, the proposed sensing platform displayed satisfactory accuracy through the validation with inductively coupled plasma-mass spectrometry (ICP-MS) and a spike-recovery analysis (recoveries, 87.92–111.61%; RSD, 4.89–8.85%), indicating the great potential for rapid and sensitive detection of Cu2+ or other metal ions.

Highly sensitive magnetic relaxation sensing method for aflatoxin B1 detection based on Au NP-assisted triple self-assembly cascade signal amplification

Highly sensitive detection of aflatoxin B1 (AFB1) is of great significance because of its high toxicity and carcinogenesis. We propose a magnetic relaxation sensing method based on gold nanoparticles (Au NPs)-assisted triple self-assembly cascade signal amplification for highly sensitive detection of AFB1. Both AFB1 antibody and initiator DNA (iDNA) are labeled on Au NPs to form Ab-Au-iDNA probe. iDNA is enriched by Au NPs to achieve first signal amplification. Different amounts of Ab-Au-iDNA were bound with AFB1 antigen by indirect competitive immunoassay, and then hybridization chain reaction event was initiated by iDNA to produce long hybridization chain reaction products to enrich more horseradish peroxidase-streptavidin for the second signal amplification. Dopamine could be rapidly converted to polydopamine by HRP catalysis, which is used as the third signal amplification. The Fe3+ solution, providing paramagnetic ions with a strong magnetic signal, could be adsorbed by the polydopamine due to the formation of coordination bonds of phenolic hydroxyl groups with Fe3+. This effective interaction between polydopamine and Fe3+ significantly changes the transverse relaxation time signal of Fe3+ supernatant solution, which can be used as a magnetic probe for highly sensitive detection of AFB1. The sensor exhibited high specificity and sensitivity with a detection limit of 0.453 pg/mL owing to the Au NP-assisted triple self-assembly cascade signal amplification strategy. It has been successfully employed for AFB1 detection in animal feed samples with consistent results of enzyme linked immune sorbent assay and high-performance liquid chromatography.

The dumbbell probe mediated triple cascade signal amplification strategy for sensitive and specific detection of uracil DNA glycosylase activity

Uracil DNA glycosylase (UDG) is a key base excision repair (BER) enzyme and its abnormal expression is nearly relevant to several diseases including cancer. The sensitive detection of UDG activity is beneficial for biomedical studies and clinic diagnosis. In this work, we proposed a dumbbell probe mediated triple cascade signal amplification strategy for sensitive and specific detection of UDG activity. The specially designed dumbbell probe contained two uracil bases, two recognition sites for nicking enzyme and a split sequence of DNAzyme. Unsealed dumbbell probes were first connected into sealed dumbbell probes by T4 DNA ligase, and then the unsealed probes were hydrolyzed by exonuclease to ensure the purity of probes. Under the influence of UDG, two uracil bases were removed to produce two apyrimidinic (AP) sites, which were subsequently cleaved by Endo.IV. The probes after cleavage acted as primers and templates for double nicking sites strand displacement amplification (SDA) to produce a mass of two products. The products of SDA continued to act as primers and templates for rolling circle amplification (RCA) to produce repeats containing complete DNAzyme sequences. The DNAzyme repeatedly cleaved multiple molecular beacons (MB), resulting in remarkable fluorescence enhancement. Benefiting from the triple cascade signal amplification, the sensitivity was improved and the detection limit was 7.2 × 10−5 U mL−1. The method could well distinguish UDG from other interfering enzymes and detect UDG activity in real biological samples, showing good specificity. In addition, this method could be used for screening inhibitors. The above results suggested that the method provided a promising analytical means for UDG related biomedical research and clinic diagnosis.

Triple signal amplification strategy for the ultrasensitive electrochemical detection of human papillomavirus 16 E6/E7 mRNA

Human papilloma virus (HPV) is the primary causative agent of cervical, vaginal, and vulvar cancers. HPV E6/E7 mRNA detection has been proven to improve the specificity and positive predictive value compared with HPV DNA testing in screening, whereby, it may possess higher diagnostic potential. Herein, to establish the ultrasensitive and specific detection of HPV E6/E7 mRNA, we developed a novel triple signal amplification strategy, combined with gold nanoparticles (AuNPs), reverse transcription loop-mediated isothermal amplification (RT-LAMP) and high affinity biotin-avidin system. This novel proposed signal amplification strategy exhibits the desired detection limit of 0.08 fM (approximately 100 copies) and a wide linear range from 0.1 pmol/mL to 100 nmol/mL for HPV16 E6/E7 mRNA detection. Importantly, the present novel biosensor is 10–100 times more sensitive than conventional RT-PCR in detecting HPV16 E6/E7 mRNA positive clinical samples. Conclusively, this biosensor shows good stability, selectivity, and reproducibility, which demonstrates its potential in future clinical diagnosis with desirable sensitivity and specificity.

A novel electrochemical biosensor for ultrasensitive Hg2+ detection via a triple signal amplification strategy.

We developed a novel electrochemical biosensor for ultrasensitive Hg2+ detection via a triple signal amplification strategy of a DNA dual cycle, organic-inorganic hybrid nanoflowers (Cu3(PO4)2 HNFs) and gold nanoparticle (AuNP) probe. The DNA dual cycle was triggered by exonuclease III (Exo III) in the presence of Hg2+, and Cu3(PO4)2 HNFs were synthesized as an AuNP probe carrier. The electrochemical biosensor displayed high stability, high sensitivity and excellent specificity, which was improved by up to seven orders of magnitude compared to the World Health Organization (WHO) allowed Hg2+ levels in drinking water. This signal amplification strategy could be easily modified and extended to detect other hazardous heavy metals and nucleic acids.

Ultrasensitive Detection of Parathyroid Hormone through Fast Silver Deposition Induced by Enzymatic Nitroso Reduction and Redox Cycling.

Enzymatically induced silver deposition and subsequent electrochemical oxidation have been widely used in electrochemical biosensors. However, this method is ineffective for producing highly enhanced silver deposition for use in ultrasensitive detection. Herein, we report a fast silver deposition method that simultaneously uses three signal amplification processes: (i) enzymatic amplification, (ii) chemical-chemical (CC) redox cycling, and (iii) chemical-enzymatic (CN) redox cycling. DT-diaphorase (DT-D) is used for enzymatic amplification to convert a nitroso compound, a species incapable of directly reducing Ag+ to an amine compound, which can directly reduce Ag+. NADH acts as a reducing agent for the indirect reduction of Ag+ via the two redox cycling processes. 4-Nitroso-1-naphthol is converted to 4-amino-1-naphthol (NH2-N) in the presence of DT-D. NH2-N initiates two redox cycling processes: NH2-N, along with Ag+ and NADH, are involved in the CC redox cycling, whereas NH2-N, along with Ag+, DT-D, and NADH, are involved in the CN redox cycling. Finally, the deposited silver is electrochemically oxidized to produce a signal. When this triple signal amplification strategy for fast silver deposition is applied to an electrochemical immunosensor for detecting parathyroid hormone (PTH), a detection limit as low as ∼100 fg/mL is obtained. The concentrations of PTH in clinical serum determined using the developed immunosensor are found to agree with those measured using a commercial instrument. Thus, the use of this strategy for fast silver deposition is highly promising for ultrasensitive electrochemical detection and biosensing applications.

Ultrasensitive analysis of carcinoembryonic antigen based on MoS2-based electrochemical immunosensor with triple signal amplification

Ultrasensitive detection of cancer biomarkers has shown great promise for precision medicine. Here, a triple signal amplification strategy was developed for analysis of carcinoembryonic antigen (CEA) by using MoS2-based nanocomposites. Gold nanoparticles-decorated molybdenum disulfide nanocomposite (MoS2-AuNPs) was used to construct the modified electrode and nanoprobe, which could efficiently amplify electrochemical signal due to its large surface area and high catalytic ability. Horseradish peroxidase (HRP)-labelled carcinoembryonic monoclonal antibody (anti-CEA) and HRP were used to co-construct the MoS2-based nanoprobe, which could further amplify the electrochemical signal by catalyzing o-phenylenediamine (o-PD) in the presence of hydrogen peroxide (H2O2). Expectedly, an excellent analytical performance for CEA detection was obtained, such as wide detection range (10 fg mL−1-1 ng mL−1), ultralow detection limit (1.2 fg mL−1), high selectivity and good stability, suggesting this immunosensor could detect CEA in real samples.

Zero background and triple-signal amplified fluorescence aptasensor for antibiotics detection in foods

It's important to eliminate matrix interference for accurate detecting antibiotic residues in complex food samples. In this study, we designed a zero-backgrounded fluorescence aptasensor to achieve on-site detection of antibiotic residues, with chloramphenicol (CAP) as representative analyte. Moreover, a three stir-bars assisted target recycling system (TSBTR) was designed to achieve triple signal amplification and increase the sensitivity. The bars included one magnetic stir-bar modified with two kinds of long DNA chains, and two gold stir-bars modified with Y shape-duplex DNA probes respectively. In the presence of CAP, the target could recurrently react with the probes on the bars and replace a large amount of long DNA chains into supernatant. After then, the bars were taken out and SYBR green dye was added to the solution. The dye can specifically intercalate into the duplex structures of DNA chains to emit fluorescence while not emitting a signal in its free state. Under the optimized experimental conditions, a wide linear response range of 5 orders of magnitude from 0.001 ng mL−1 to 10 ng mL−1 was achieved with a detection limit of 0.033 pg mL−1 CAP. The assay was successfully employed to detect CAP in food samples (milk & fish) with consistent results with ELISA's. High selectivity and sensitivity were attributed to the zero background signal and triple signal-amplification strategy. Moreover, the detection time can be shortened to 40 min due to that three signal amplified process can occur simultaneously. The fluorescent aptasensor was also label- and enzyme-free. All these ensure the platform to be rapid, cost-effective, easily-used, and is especially appropriate for detection antibiotics in food safety.

A novel electrochemical strategy based on porous 3D graphene-starch architecture and silver deposition for ultrasensitive detection of neuron-specific enolase.

This work was aimed at designing a novel and ultrasensitive electrochemical immunoassay strategy to detect neuron-specific enolase (NSE) with a triple signal amplification strategy. A greatly enhanced sensitive detection of NSE was achieved by using porous three-dimensional graphene-starch architecture (3D-GNS) modified on the immunosensor's surface to construct a unique 3D immunoelectrode, which would greatly accelerate electron transfer and capture more protein molecules. 3D-GNS was prepared with starch as a crosslinking agent and stabilizer, which is biocompatible and environmentally friendly. Aggregation-free gold nanoparticle (AuNP) incorporated ordered mesoporous carbon-silica (OMCSi-Au) with good catalytic activity was synthesized as the tracing tag for labeling signal antibody (Ab2). After a sandwich-type immunoreaction, the OMCSi-Au labeled Ab2 was trapped on the surface of the immunosensor, and the high concentration of AuNPs with high dispersion greatly catalyzed the deposition of silver nanoparticles. The deposited silver nanoparticles (AgNPs) could be tested directly with anodic stripping voltammetric analysis (ASV) in potassium chloride solution to monitor the immunoreactions, which greatly enhanced the sensitivity of protein markers with a detection limit of 0.008 pg mL-1, and a linear range of 0.02 pg mL-1 to 35 ng mL-1 for neuron-specific enolase antigen. This proposed immunosensor displayed acceptable accuracy, good stability, and high sensitivity. Good results were also obtained in agreement with the enzyme-linked immunosorbent assay method, which provides greatly promising potential in clinical applications.

Triple Signal Amplification Strategy for Ultrasensitive Determination of miRNA Based on Duplex Specific Nuclease and Bridge DNA-Gold Nanoparticles.

Abnormal expression of miRNAs always occurs in solid tumors. Thus, it is critical to sensitively and selectively detect such biomarkers for the diagnosis and prognosis of diseases. Here, we report a biosensing scheme for the determination of miRNA with triple signal amplification based on target-triggered cyclic duplex specific nuclease digestion and bridge DNA-gold nanoparticles. Electrochemical signals are recorded to present initial levels of miRNA. This method is ultrasensitive with a wide linear range of 10-17 to 10-11 M. The limit of detection is down to 6.8 aM. Moreover, the overexpression of miR-21 is confirmed in lung cancer patients by the proposed method, which is in good accordance with qRT-PCR results. In addition, the developed biosensor does not need a reverse transcription process or any thermal cycling processes. Its performance satisfies the requirement for convenient, rapid, sensitive, and specific early diagnosis of cancers. Therefore, it may have great potential utility in the near future.

Sensitive determination of sialic acid expression on living cells by using an ITO electrode modified with graphene, gold nanoparticles and thionine for triple signal amplification

The authors demonstrate a multivalent recognition and triple signal amplification strategy for amperometric cytosensing and dynamic evaluation of cell surface sialic acid (SA). The strategy is based on the use of (a) graphene oxide functionalized with 3-aminophenylboronic acid (APBA-CG) for cell capture, and (b) of Sambucus nigra agglutinin and thionine conjugated to gold nanoparticles (SNA-AuNP-Th) acting as a nanoprobe. The use of APBA-CG accelerates electron transfer and warrant a large surface area for cell capture. The AuNPs, in turn, nanoprobe provide an effective matrix for mass loading of thionine molecules and thus dramatically improve sensitivity. The results obtained with the biosensor demonstrate that sialic acid is strongly expressed levels in cancer cells compared to normal ones. The cytosensor was applied to the detection of HepG2 cells. Best operated at a working voltage of typically 0.42 V vs. Ag/AgCl, it displays a wide linear range (that extends from 1.0 × 102 to 1.0 × 107 cells per mL and a low detection limit (50 cells per mL)). The method was successfully applied dynamic monitoring of the changes in cell surface SA expression in response to the addition of the enzyme sialidase. In our perception, the SNA-based biosensor represents a viable tool to study SA-related biological processes and for diagnosis and treatment of early stages of cancer.

Ultrasensitive amperometric immunosensor for PSA detection based on Cu2O@CeO2-Au nanocomposites as integrated triple signal amplification strategy

In this work, a novel label-free electrochemical immunosensor was developed for the quantitative detection of prostate specific antigen (PSA). To this end, the amino functionalized cuprous oxide @ ceric dioxide (Cu2O@CeO2-NH2) core-shell nanocomposites were prepared to bond gold nanoparticles (Au NPs) by constructing stable Au-N bond between Au NPs and -NH2. Because the synergetic effect presents in Cu2O@CeO2 core-shell loaded with Au NPs (Cu2O@CeO2-Au), it shows better electrocatalytic activity towards the reduction of hydrogen peroxide (H2O2) than single Cu2O, Au NPs and Cu2O@CeO2. Featured by large specific surface area, good biocompatibility and good electrochemical properties which can greatly improve the electronic transmission rate, Cu2O@CeO2-Au was used as transducing materials to achieve efficiently capture antibodies and triple signal amplification of the proposed immunosensor. Under the optimal conditions, the proposed immunosensor exhibited a wide linear range from 0.1pg/mL to 100ng/mL with a low detection limit of 0.03pg/mL (S/N=3). Furthermore, the proposed label-free immunosensor has been used to determine PSA in human serum with satisfactory results. Meanwhile, it displayed good reproducibility, acceptable selectivity, and long-term stability, which had promising application in bioassay analysis.

Triple signal amplification strategy based on size and shape transformation of ultrasmall sub-10 nm gold nanoparticles tag towards sensitivity improvement of electrochemical immunosensors

Triple signal amplification strategy was designed to enhance the electrochemical detection sensitivity of immunosensing platform based on gold nanoparticles (AuNP) tagging. The strategy described herein includes the stepwise enlargement of AuNP tag. The first step was achieved by reducing gold chloride ion in presence of AuNP acting as nucleation seeds. In this step, size of AuNP was increased by the growth of gold shell over small AuNP tag. The second step was accomplished by initiating the growth of spiky AuNP. This particular step will offer a large increase of AuNP size and surface area due to the change of nanoparticles shape. Finally, the silver enhancement step was used to provide an electroactive layer for electrochemical detection. The sandwich immunoassay was carried out using prostate specific antigen (PSA), as a model analyte. Precursors and enhancement process, which were commonly used for spiky AuNP synthesis, were adapted and optimized to achieve the highest electrochemical signal. Since the spiky AuNP enhancement step is established and reported here for the first time, the effect of relevant experimental conditions, i.e. concentration and ratio of Au and Ag ions, reducing agents, and reaction time, were thoroughly examined. Size of AuNP tag and enhancing steps were also confirmed to have the most influence on the successful enhancement process. By understanding the valuable insight of this approach, detection sensitivity of applicable AuNP-tagged immunoassay can be significantly improved. Based on an electrochemical immunosensor demonstrated here, the triple signal amplification leads to over 260-fold of signal increase in comparison with conventional silver enhancement process. Therefore, this approach would be promising strategy for improving detection limit of AuNP-tagged sensing platform.