Recycling Amplification Research Articles

In-situ cation-exchange deposited Ag2S/In4SnS8 of Z-type heterojunction coupled with DNA recycling amplification for photoelectrochemical biosensing

Herein, a novel photoelectrochemical (PEC) biosensor was developed utilizing an in situ cation-exchange method to deposit a high-performance Ag2S/In4SnS8, a Z-type heterojunction with co-shared S atoms, as a signal indicator and using DNA recycling amplification for the sensitive and accurate detection of miRNA-141. The photosensitive Ag2S deposited on the In4SnS8 surface not only facilitated the rapid generation of photoelectrons but also formed a Z-type heterojunction with In4SnS8, effectively inhibiting electron-hole pair recombination. This resulted in a considerable improvement in the photoelectric conversion efficiency of Ag2S/In4SnS8, yielding an ∼ 120-fold increase in photocurrent compared to that of In4SnS8 under 660-nm visible-light irradiation. Furthermore, the arborescent DNA structure formed by hybrid chain reaction (HCR) enhanced the steric effect, thereby reducing the PEC response. Importantly, numerous quenchers MnPP competed for light absorption and captured photogenerated electrons in the valence bands of In4SnS8 and Ag2S, reducing electron transfer to the electrode and further considerably reducing the photocurrent. This enabled the sensitive detection of miRNA-141 across a concentration range from 10 amol·L−1 to 100 pmol·L−1, with a limit of detection as low as 3.3 amol·L−1 (S/N = 3). The proposed biosensor exhibits excellent stability and is expected to be applied for detecting clinically relevant disease markers.

Dual-signal biosensor based on G-quadruplex/hemin DNAzyme and zinc-doped molybdenum disulfide quantum dots for ultrasensitive detection of tumor biomarker

Herein, a dual-mode fluorometric and colorimetric biosensor for Pax-5a gene was developed based on zinc-doped molybdenum disulfide quantum dots (Zn-MoS2 QDs) by coupling exonuclease-assisted recycling amplification and peroxidase-mimic DNAzyme. In the presence of Pax-5a gene, the exonuclease III can cleave the duplexes formed by Pax-5a gene and the hairpin DNA (HP), releasing the output DNA (oDNA). G-rich DNA and magnetic beads (MBs) labeled with capture DNA (cDNA) can hybridize with oDNA to form the MBs-cDNA/oDNA/G-rich DNA sandwich complex. The remaining G-rich DNA in the supernatant through magnetic separation could bind hemin to produce G-quadruplex/hemin peroxidase-mimicking DNAzyme, which catalyzed the oxidization of 3,3'-diaminobenzidine (DAB) by H2O2. The generated brown oxidation product (oxDAB) had a distinct absorption peak at 464 nm and could quench the fluorescence of Zn-MoS2 QDs at 406 nm. The high peroxidase activity of DNAzyme, recycling amplification strategy and magnetic separation technique led to excellent sensitivity and specificity of this detection platform. The detection limits of Pax-5a gene by fluorometric and colorimetric methods were 0.52 pM and 1.12 pM, respectively. Furthermore, this sensing system was successfully applied to Pax-5a gene determination in human serum samples, which had promising potential in biochemical analysis and clinical diagnosis.

Construction of an efficient microRNA sensing platform based on terminal deoxynucleotidyl transferase-mediated synthesis of copper nanoclusters

A conceptual innovative electrochemical sensing platform was constructed for microRNA (miRNA) detection based on terminal deoxynucleotidyl transferase (TdT)-mediated synthesis of copper nanoclusters. In principle, the substrate strand immobilized on working electrode acted as the template for the TdT-mediated DNA extension reaction, while the DNAzyme probe would be activated in the absence of miRNA 21, which led to the cleavage of the substrate strand to produce the 5’ phosphate terminus and inhibited the TdT-mediated DNA extension reaction. On the other hand, the presence of miRNA 21 initiated the toehold-mediated strand displacement reaction, causing the disassembly of the DNAzyme structure and preventing the substrate strand from being cleaved. This enabled the recycle amplification of target miRNA 21, and TdT catalyzed the DNA extension reaction on the 3’ hydroxyl terminus of the substrate strand, producing a large number of poly thymine (polyT) sequences. These sequences bound with copper ions for in-situ synthesis of copper nanoclusters on the sensing electrode, and the increased electrochemical signal of copper was recorded by differential pulse stripping voltammetry (DPSV), which paved the way for sensitive determination of miRNA 21 extracted from cancer cells with a detection limit of 36 aM. Due to its high sensitivity, user-friendly operation, and cost-effectiveness, this method is helpful for the fundamental research of sensing mechanism as well as the diagnosis of related diseases.

Electrochemiluminescence Biosensor for Ascorbic Acid Based on Target Transformation of Cell-Free RNA Transcription System and Duplex-Specific Nuclease-Assisted Recycling Amplification.

A cell-free RNA transcription system had been coupled with electrochemiluminescence (ECL) detection technology for the first time to develop an ascorbic acid (AA, acting as a model target) biosensor. The biosensor is composed of single-stranded DNA (ssDNA) sequences modified with alkynyl and azido groups, respectively, alongside an incomplete gene circuit framework. The addition of target AA and copper ions will cause the linkage of the two ssDNA sequences through a click chemistry reaction. This results in the subsequent reconstruction of a complete gene circuit. The reconstituted gene circuit, in conjunction with the T7 RNA polymerase, drives the transcription of substantial quantities of RNA. ssDNA labeled with ferrocene (Fc) (Fc-DNA) had been immobilized on a tris(2,2'-bipyridyl) ruthenium(II) chloride hexahydrate-doped SiO2 nanoparticle (Ru@SiO2 NPs) modified electrode first. The quenching effect of Fc on Ru@SiO2 causes the low ECL detected. The transcribed RNA sequence assisted double-stranded specific nuclease (DSN) to cut the ssDNA-Fc and the ECL of the system was enhanced. Optimal experimental conditions reveal that the ECL signal exhibits a linear correlation with the logarithmic concentration of AA, spanning a detection range from 100 nM to 1 mM, with a detection limit of 45 nM. This innovative methodology expands the utility of a cell-free RNA transcription system within the realm of biosensing applications.

A fluorescence-electrochemical dual-mode aptasensor for ultrasensitive detection of lead based on exonuclease-catalyzed recycling and hybridization chain reaction amplification

To improve the accuracy and reliability of the detection system, this study combined the exonuclease-assisted autocatalytic target cycling fluorescence strategy with an electrochemical platform to establish a dual-mode fluorescence-electrochemical aptasensor for the ultra-sensitive quantification of lead ion (Pb2+). In the fluorescence detection strategy, ssDNA could hybridize with the aptamer to form double-stranded DNA. With the addition of Pb2+, the aptamer dissociated from the double-stranded DNA to capture Pb2+ and trigger the enzymatic digestion process, leading to the target recirculation to achieve the first signal amplification. The free ssDNA could then hybridize with S1, and the released S2 was able to trigger a hybridization chain reaction (HCR) in the electrochemical system, achieving the second signal amplification. The quantification of Pb2+ was realized by measuring both the fluorescence and the differential pulse voltammetry signals with detection limits of 0.028ng/mL and 0.788pg/mL, respectively. This fluorescence-electrochemical dual-mode aptasensor overcame the limitations of complex matrices and environmental interference, and provided a valuable reference for the development of reliable multi-signal sensors in water, tea and vegetable safety monitoring.

Immobilization coupling with aptamer assisted dual cycle amplification for sensitive sEVs isolation and analysis.

Precise identification of small extracellular vesicles (sEVs) is crucial for improving disease diagnosis and treatments, such as bladder cancer. However, accurate isolation and simultaneously quantification of sEVs remain a huge challenge. We have introduced a new technique that combines immobilization with aptamer-assisted dual cycle amplification to isolate and analyze sEVs with high sensitivity. In this method, the CD9 protein antibody is attached to the plate's surface for the initial identification of sEVs, while an aptamer probe is used to detect the exosomal surface protein CD63. We have created an sEVs-surface method that combines target recognition initiated signal recycling and rolling circle amplification (RCA) for signal amplification. This approach allows for the "AND" logic analysis of dual biomarkers, enabling both sEVs quantification and tracing. The proposed approach has a broad detection range and a low limit of detection. Moreover, the established method showed good stability in detecting sEVs with a low coefficient of variation. Our method can effectively isolate certain sEVs and accurately identify them, making it suitable for many uses in biological science, biomedical engineering, and personalized medicine.

A Highly Sensitive Electrochemical Aptasensor for Kanamycin: Leveraging RecJf Exonuclease-Assisted Target Recycling and Hybridization Chain Reaction Signal Amplification

A Highly Sensitive Electrochemical Aptasensor for Kanamycin: Leveraging RecJf Exonuclease-Assisted Target Recycling and Hybridization Chain Reaction Signal Amplification

A sensitive tobramycin electrochemical aptasensor based on multiple signal amplification cascades

The residue of tobramycin, a broad spectrum antibiotic commonly used in animal husbandry, has evitable impact on human health, which may cause kidney damage, respiratory paralysis, neuromuscular blockade and cross-allergy in humans. Sensitive monitoring of tobramycin in animal-derived food products is therefore of great importance. Herein, a new aptamer electrochemical biosensor for sensing tobramycin with high sensitivity is demonstrated via exonuclease III (Exo III) and metal ion-dependent DNAzyme recycling and hybridization chain reaction (HCR) signal amplification cascades. Tobramycin analyte binds aptamer-containing hairpin probe to switch its conformation to expose the toehold sequence, which triggers Exo III-based catalytic digestion of the secondary hairpin to release many DNAzyme strands. The substrate hairpins immobilized on the Au electrode (AuE) are then cyclically cleaved by the DNAzymes to form ssDNAs, which further initiate HCR formation of lots of long methylene blue (MB)-tagged dsDNA polymers on the AuE. Subsequently electro-oxidation of these MB labels thus exhibit highly enhanced currents for sensing tobramycin within the 5–1000 nM concentration range with an impressive detection limit of 3.51 nM. Furthermore, this strategy has high selectivity for detecting tobramycin in milk and shows promising potential for detect other antibiotics for food safety monitoring.

Fluorometric determination of mecA gene in MRSA with a graphene-oxide based bioassay using flap endonuclease 1-assisted target recycling and Klenow fragment-triggered signal amplification

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant bacterium that causes a wide range of illnesses, necessitating the development of new technologies for its detection. Herein, we propose a graphene oxide (GO)-based sensing platform for the detection of mecA gene in MRSA using flap endonuclease 1 (FEN1)-assisted target recycling and Klenow fragment (KF)-triggered signal amplification. Without the target, all the DNA probes were adsorbed onto GO, resulting in fluorescence quenching of the dye. Upon the addition of the target, a triple complex was formed that triggered FEN1-assisted target recycling and initiated two polymerization reactions with the assistance of KF polymerase, generating numerous dsDNA that were repelled by GO. These dsDNAs triggered fluorescence enhancement when SYBR Green I was added. Therefore, the target DNA was quantified by measuring the fluorescence at excitation and emission wavelengths of 480/526 nm. This mecA gene assay showed a good linear range from 1 to 50 nM with a lower limit of detection of 0.26 nM, and displayed good applicability to the analysis of real samples. Thus, a new method for monitoring MRSA has been developed that has great potential for early clinical diagnosis and treatment.

An ultra-sensitive electrochemical biosensor for circulating tumor DNA utilizing dual enzyme-assisted target recycle and hybridization chain reaction amplification strategies

Circulating tumor DNA (ctDNA) has emerged as a pivotal biomarker for cancers, offering a non-invasive means of detection through liquid biopsies. However, the current methodologies for ctDNA detection often struggle with sensitivity, especially when confronted with ultra-low concentrations. To address this challenge, we have engineered a remarkably sensitive electrochemical biosensor that can detect low concentration of ctDNA. This breakthrough is achieved by integrating a dual enzyme-assisted amplification mechanism with hybridization chain reaction (HCR). In the initial phase, the presence of ctDNA triggers the unfolding of the H2 hairpin structure, resulting in a segment of double-stranded DNA. The subsequent introduction of Klenow (3′→5′ exo-) and nicking endonuclease (Nb.BbvCI) enzymes leads to the abundant production of extended DNA (EXTDNA). This step critically enhances the initial amplification of the target DNA, ensuring superior specificity and selectivity thanks to the synergistic enzymatic activity. EXTDNA efficiently unfolds the H1 hairpin structure immobilized on the gold electrode, which acts as the initiation point for the HCR. Further amplification of the trace target DNA is achieved through an additional H3 and H4 hybrid-mediated HCR. Finally, the differential pulse voltammetry signal of methylene blue is increased significantly. Our results reveal that the developed electrochemical biosensor displays an exceptional linear correlation with ctDNA across concentrations ranging from 10 fM to 20 pM, with an unprecedented detection limit of 2.3 fM (3 s/k). This innovative approach shows immense potential for the ultrasensitive detection of ctDNA, promising broad applications in early cancer detection and monitoring.

An electrochemical biosensor with homogeneous dual amplification strategy for sensitive analysis of Pb2+

Herein, a homogeneous double amplified electrochemical biosensing system was designed on the basis of rolling circle amplification (RCA) and DNAzyme-assisted Pb2+ recycling amplification with the assistance of the signal molecule methylene blue (MB), which was used for highly sensitive analysis of Pb2+. In comparison with the conventional electrochemical biosensor, the homogeneous strategy adopted in this work can avoid the complex electrode modification and immobilization processes, and address the issues of the large steric hindrance and the restricted probe freedom on the sensing interface, thus achieving high recognition efficiency between recognition unit and target molecule as well as excellent detection performance of biosensor. In addition, the double amplified method assembling RCA and DNAzyme-assisted Pb2+ recycling amplification can convert a handful of target Pb2+ into abundant signal molecule MB, which would lead to the double amplification of output signal and the significantly enhanced detection sensitivity. Taking advantages of homogeneous dual amplification strategy, the constructed electrochemical biosensor for Pb2+ analysis emerged a good linear range of 50 fM to 500 nM and a detection limit of 16.7 fM with significant selectivity and desirable stability. Impressively, the result presented herein comprise a valuable reference for establishing homogeneous multiple amplification model and constructing innovative biosensing system for sensitive detection of heavy metal ions.

A highly sensitive fluorescence biosensor for aflatoxins B1 detection based on polydiacetylene liposomes combined with exonuclease III-assisted recycling amplification.

Afluorescence biosensor for determination of aflatoxin B1 (AFB1)based on polydiacetylene (PDA) liposomes and exonuclease III (EXO III)-assisted recycling amplification was developed. The AFB1 aptamer partially hybridizes with complementary DNA (cDNA), which is released upon recognition of AFB1 by the aptamer. Subsequently, the cDNA hybridizes with hairpin H to form double-stranded DNA that undergoes digestion by EXO III, resulting in the cyclic release of cDNA and generation of capture DNA for further reaction. The capture DNA then hybridizes with probe modified on PDA liposomes, leading to aggregation of liposomes and subsequent fluorescence production. This strategy exhibited a limit of detection of 0.18 ng/mL within the linear range 1-100 ng/mL with adetermination coefficient > 0.99. The recovery ranged from 92.81 to 106.45%, with relative standard deviations (RSD) between 1.73 and 4.26%, for corn, brown rice, peanut butter, and wheatsamples. The stability, accuracy, and specificity of the method demonstrated the applicability for realsampleanalysis.

Amplified Assembly of G-Quadruplex-Decorated DNA Network Nanostructure toward AIE Signaling-Based Sensitive Biosensing.

Aggregation-induced emission (AIE) has offered a promising approach for developing low-background fluorescent methods; however, its applications often suffer from complex probe synthesis and poor biocompatibility. Herein, a novel AIE biosensing method for kanamycin antibiotic assays was developed by utilizing a DNA network nanostructure assembled from an aptamer recognition reaction to capture a large number of tetraphenylethylene fluorogen-labeled signal DNA (DTPE) probes. Due to the excellent hydrophilicity of the oligonucleotides, DTPE exhibited excellent water solubility without obvious background signal emission. Based on an ingenious nucleotide design, an abundance of G-quadruplex blocks neighboring the captured DTPE were formed on the DNA nanostructure. Because of the greatly restricted free motion of DTPE by this unique nanostructure, a strong AIE fluorescence signal response was produced to construct the signal transduction strategy. Together with target recycling and rolling circle amplification-based cascade nucleic acid amplification, this method exhibited a wide linear range from 75 fg mL-1 to 1 ng mL-1 and a detection limit down to 24 fg mL-1. The excellent analytical performance and effective manipulation improvement of the method over previous approaches determine its promising potential for various applications.

Novel Enzyme-Assisted Recycle Amplification Strategy for Tetracycline Detection Based on Oxidized Single-Walled Carbon Nanohorns.

In this study, oxidized single-walled carbon nanohorns (oxSWCNHs) were prepared using nitric acid oxidation and subsequently combined with 3'6-carboxyfluorescein through charge transfer to prepare fluorescent probes. These oxSWCNHs were used to quench fluorogen signals at short distances and dissociate ssDNA using cryonase enzymes. We established a method for rapidly detecting tetracycline (TC) in complex samples based on the amplification of cryonase enzyme signals. After optimizing the experimental conditions, our method showed a detection limit of 5.05 ng/mL, with good specificity. This method was used to determine the TC content in complex samples, yielding a recovery rate of 90.0-103.3%. This result validated the efficacy of our method in detecting TC content within complex samples.

Molecularly imprinted polymer combined with MOF-assisted redox recycling amplification: A powerful electrochemical sensing strategy for pathogenic bacteria

Rapid and sensitive determination of foodborne pathogenic bacteria at a low cost is crucial for ensuring food safety monitoring and diagnosing bacterial infections. In this protocol, we propose an electrochemical sandwich-type biosensor for highly sensitive detection of bacteria based on a bacterial imprinted polymer film (BIF) and metal-organic frameworks (MOF)-assisted redox recycling amplification. The BIF is formed in-situ through electropolymerization (EP) and template removal on the electrode surface, while aptamers are assembled on nanogold-modified MOF to create signal nanoprobes with target recognition ability. The BIF-modified electrode effectively captures the target bacteria and subsequently forms a sandwich structure through conjugation with signal nanoprobes, enabling efficient adsorption of methylene blue (MB). This facilitates the redox recycling of Fe(II) from the electroactive [Fe(CN)6]3-/4- in testing buffer assisted by MB, thereby generating a significantly amplified anodic current of [Fe(CN)6]3-/4- for precise quantitation. By utilizing the selective BIF interface and MOF-assisted redox recycling amplification, our method demonstrates excellent analytical performance with a low limit of detection (LOD) of 1 CFU mL−1, a broad detection range spanning from 10 to 108 CFU mL−1, and high selectivity. The feasibility of applying this approach for detecting bacteria in complex samples highlights its significant potential in areas related to food safety and public health.

Microfluidic bead-based biosensor: Ultrasensitive ctDNA detection based on duplex-functional split-DNAzyme and dendritic enzyme-free signal amplification

Circulating tumor DNA (ctDNA) is a crucial cancer biomarker for early or noninvasive monitoring, which is essential for developing ultrasensitive and selective assays in cancer diagnosis and treatment. Herein, a cascade signal amplification of duplex-functional split-DNAzyme and dendritic probes was proposed for ultrasensitive and specific detection of nasopharyngeal carcinoma-associated Epstein-Barr virus (EBV) DNA on microfluidic microbead array chips. With the assistance of Pb2+, the duplex-functional split-DNAzyme recognizes EBV DNA and then rapidly cleaves the substrate strand. Subsequently, the released target could be recycled, and its exposed capture probe, triggered the dendritic enzyme-free signal amplification. As the enhanced mass transfer capability, target recycling, and dendritic DNA structure signal amplification inherent to microfluidic bead arrays were integrated, it achieved an excellent detection limit of 0.36 fM and a wide linear range of 1 fM∼103 fM. Further, it was applied to content detect simulated samples of EBV DNA, recovery ranged from 97.2 % to 108.1 %, and relative standard deviation (RSD) from 3.3 % to 5.9 %, exhibiting satisfactory recovery results. The developed microfluidic biosensor was a high-sensitivity and anti-interference system for ctDNA analysis, with minimal reagent volumes (microlitres) required. Thus, it is a promising platform for ctDNA at the lowest level at their earliest incidence.

A cascade signal-amplified fluorescent biosensor combining APE1 enzyme cleavage-assisted target cycling with rolling circle amplification.

A cascade signal-amplified fluorescent biosensor was developed for miRNA-21 detection by combining APE1 enzyme-assisted target recycling and rolling circle amplification strategy. A key feature of this biosensor is its dual-trigger mechanism, utilizing both tumor-endogenous miRNA-21 and the APE1 enzyme in the initial amplification step, followed by a second rolling circle amplification reaction. This dual signal amplification cascade significantly enhanced sensitivity, achieving a detection limit of 3.33 pM. Furthermore, this biosensor exhibited excellent specificity and resistance to interference, allowing it to effectively distinguish and detect the target miRNA-21 in the presence of multiple interfering miRNAs. Moreover, the biosensor maintained its robust detection capabilities in a 10% serum environment, demonstrating its potential for clinical disease diagnosis applications.

Cascaded recycling amplification mediated in situ synthesis of silver nanoclusters for the construction of sensitive and label-free electrochemical sensor

A growing number of studies have shown the crucial role of microRNA (miRNA) sensing in cancer clinical diagnosis and prognosis research. In this study, a label-free and enzyme-free electrochemical sensor was presented to achieve sensitive determination of miRNA 122, which was constructed based on in situ synthesis of silver nanoclusters (AgNCs) mediated by cascaded recycling amplification. The efficient and cascaded recycling amplification was initiated by the target miRNA 122, resulting in the release of signal probe with rich cytosine bases, which would be loaded on the sensing interface for in situ synthesis of silver nanoclusters, leading to dramatically increased current for quantitative analysis. This method achieved selective and robust miRNA 122 sensing in human serum samples with a detection limit down to 27 aM. The proposed work would provide an innovative method for determining different biomarkers, holding great potential for boosting the development of clinical diagnosis and drug investigation.

Ratiometric Electrochemiluminescence of Zirconium Metal-Organic Framework as a Single Luminophore for Sensitive Detection of HPV-16 DNA.

This study developed a new zirconium metal-organic framework (MOF) luminophore named Zr-DPA@TCPP with dual-emission electrochemiluminescence (ECL) characteristics at a resolved potential. First, Zr-DPA@TCPP with a core-shell structure was effectively synthesized through the self-assembly of 9,10-di(p-carboxyphenyl)anthracene (DPA) and 5,10,15,20-tetra(4-carboxyphenyl)porphyrin (TCPP) as the respective organic ligands and the Zr cluster as the metal node. The reasonable integration of the two organic ligands DPA and TCPP with ECL properties into a single monomer, Zr-DPA@TCPP, successfully exhibited synchronous anodic and cathodic ECL signals. Besides, due to the impressively unique property of ferrocene (Fc), which can quench the anodic ECL but cannot affect the cathodic ECL signal, the ratiometric ECL biosensor was cleverly designed by using the cathode signal as an internal reference. Thus, combined with DNA recycle amplification reactions, the ECL biosensor realized sensitive ratiometric detection of HPV-16 DNA with the linear range of 1 fM-100 pM and the limit of detection (LOD) of 596 aM. The distinctive dual-emission properties of Zr-DPA@TCPP provided a new idea for the development of ECL luminophores and opened up an innovative avenue of fabricating the ratiometric ECL platform.

Simultaneous detection of multiplex biomarkers related with hepatocellular carcinoma by coupling DNase I-assisted recycling amplification and microfluidic electrokinetic stacking chip with parallel multi-channels

Hepatocellular cancer (HCC) is the fourth most common death related cancer in worldwide. Given that simultaneous multiplex biomarker detection might significantly increase the sensitivity and specificity of HCC diagnosis. Herein, we developed a strategy by coupling 6-carboxyfluorescein labelled aptamer (FAM-Apt), reduced graphene oxide (rGO), deoxyribonuclease I (DNase I)-assisted recycling amplification and microfluidic electrokinetic stacking chip (FAM-Apt/rGO/DNase I-MESC) for detecting multiple HCC biomarkers including α-fetoprotein (AFP), carcinoembryonic antigen (CEA) and microRNA-21 (miR-21). Thereinto, FAM-Apts were first adsorbed by rGO, resulting in fluorescence quenching, and then the FAM-Apts dissociated from rGO after binding the target. Subsequently, the FAM-Apts were degraded by DNase I and the targets were released and recognized by the FAM-Apts on the surface of rGO again. The cyclic dissociation and degradation of FAM-Apts produced FAMs and achieved primary signal amplification. Within 30 min, the released FAMs were accumulated and detected in MESC with three parallel micro-channels, resulting in secondary signal amplification. Based on FAM-Apt/rGO/DNase I-MESC, the limit of detection (LOD) for AFP, CEA and miR-21 were 37.0 pg/mL, 4.5 pg/mL and 1.3 fM, respectively. Remarkably, our method was successfully applied to detect AFP, CEA, and miR-21 in clinical serum samples. The levels of AFP and CEA achieved by our method had a good agreement with the enzyme-linked immunosorbent assay (ELISA) results. The sensitivities of AFP, CEA, miR-21 and the combination of the three targets for differentiating HCC patients from healthy individuals were 71.43%, 57.14%,85.71% and 100%, respectively, indicating the significance of the simultaneous detection of multiple indicators in clinic.