Detection Of Low-abundance Biomarkers Research Articles

Development of dual-mode ELISA based on ALP-catalyzed APP hydrolysis for IL-6 detection

Sensitive and accurate detection of interleukin 6 (IL-6) is crucial for the early diagnosis of cerebral infarction to improve patient survival rates. However, the low-abundance of IL-6 in cerebral infarction presents a significant challenge in developing effective diagnosis method. Herein, we studied and analyzed the strong fluorescence property of 4-aminophenol phosphate (APP) and developed an enzyme-linked immunosorbent assay (ELISA) for IL-6 detection. The detection was based on the integration of optical signal change induced by alkaline phosphatase (ALP)-catalyzed APP hydrolysis and ALP-mediated ELISA. The generated colorimetric signal of 4-aminophenol, APP hydrolysis product, was used for ELISA of IL-6 with a detection limit of 0.1 ng/mL, and the visual detection of IL-6 was achieved. The changes in APP fluorescence have a good linear relationship with the logarithm of IL-6 concentration in the range of 0.005 ng/mL to 5.0 ng/mL, with a detection limit of 0.001 ng/mL, which was 100 times lower than that of conventional pNPP-based ELISA. Furthermore, the constructed ELISA effectively distinguished between samples from patients with cerebral infarction and volunteers with non-cerebral infarction, and the severity of symptoms was well distinguished based on IL-6 measurement. The dual-mode ELISA demonstrated high feasibility of low-abundance biomarker detection and displayed good potential for accurate in vitro diagnosis.

Spherical nucleic acid enzyme programmed network to accelerate CRISPR assays for electrochemiluminescence biosensing applications

Given the targeted binding ability and cleavage activity of the emerging CRISPR/Cas12a assay which transduces the target into its cleavage activity exhibited broadly prospective applications in integrated sensing and actuating system. Here, we elaborated a universal approach to quickly activate CRISPR/Cas12a for low-abundance biomarker detection based on the amplification strategy of a target-induced spherical nucleic acid enzyme (SNAzyme) network that could accelerate the output of activators. Specifically, multifunctional Y-shaped probes and hairpin probes (HPs, which contained the specific sequence of the activators of CRISPR/Cas12a and the substrate chain of DNAzyme) were rationally designed to construct SNAzyme. Target recognition induced disassembly of the Y-shaped probes, which released DNAzyme strands to active DNAzyme and accompanied by SNAzyme self-assembly into SNAzyme network. Interestingly, compared with randomly dispersed SNAzyme, the reaction kinetics of the SNAzyme network enhanced 1.6 times in response to Α-methyl acyl-CoA racemase (AMACR, a biomarker for prostate cancer), which was attributed to the promoted catalytic efficiency of DNAzyme by the confined SNAzyme network. Benefiting from these, the prepared biosensor based on electrochemiluminescence (ECL) platform by loading AuAg nanoclusters (AuAgNCs) into metal-organic framework-5 (MOF-5) exhibited satisfying detection performance for AMACR with a wide linear range (0.001 μg/mL to 100 μg/mL) and a low detection limit (1.0 × 10−4 μg/mL, which exhibited significant potential in clinical diagnoses.

Tannic acid-accelerated fenton chemical reaction amplification for fluorescent biosensing: The proof-of-concept towards ultrasensitive detection of DNA methylation

As a promising biomarker, the level of methylated DNA usually changes in the early stage of the cancer. Ultrasensitive detection of the changes of methylated DNA offers possibility for early diagnosis of cancer. In this work, a tannic acid-accelerated Fenton chemical reaction amplification was firstly proposed for the construction of ultrasensitive fluorescent assay. Tannic acid was used as reductant to accelerate Fenton reaction procedure through the conversion of Fe3+/Fe2+, generating hydroxyl radicals (·OH) continuously. The produced ·OH oxidized massive non-fluorescent terephthalic acid (TA) to fluorescent-emitting hydroxy terephthalic acid (TAOH). In this way, the fluorescent signal could be greatly enhanced and the sensitivity was improved almost 116 times. The proposed signal amplification strategy was further applied to detect of DNA methylation with the assistance of liposome encapsulated with tannic-Fe3+ complexes. The methylated DNA was firstly captured through the hybridization with its complementary DNA that were pre-modified in the 96-well plate via the combination between streptavidin (SA) and biotin. Then, 5 mC antibody on the surface of liposomes specially recognized and combined with methylation sites, which brought large amount of tannic-Fe3+ complexes to participate Fenton reaction. The fluorescence of generated TAOH was depended on the concentration of methylated DNA. The assay showed good analytical performance for methylated DNA with a limit of detection (LOD) of 1.4 fM. It's believed that tannic acid-accelerated Fenton chemical reaction amplification strategy provides a promising platform for ultrasensitive fluorescent detection of low abundant biomarkers.

Photoactive hydrogels for pre-concentration, labelling, and controlled release of proteins.

We report a novel hydrogel for pre-concentration, fluorescent labelling, and light-triggered release of proteins for detection of low abundance biomarkers. The hydrogel was a co-polymer of acrylamide/bisacrylamide and methacrylamide attached to fluorescein isothiocyanate via a light cleavable bond and a poly(ethylene glycol) spacer arm of molecular weight of 3400 g mol-1. Unlike previous work, proteins were captured by an irreversible chemical reaction rather than by non-covalent affinity binding or physical entrapment. Because the protein-reactive group was attached to fluorescein, which in turn was coupled to the hydrogel by a photocleavable bond, on release the protein was labelled with fluorescein. Our hydrogel offered a pre-concentration factor of up to 236 for a model protein, streptavidin. Each protein molecule was labelled with 85 fluorescein molecules, and 50% of the proteins in the hydrogel were released after UV exposure for ∼100 s. The proteins released from the hydrogel were captured in biotinylated microtitre plates and detected by fluorescence, allowing measurement of at least 0.01 ppm (or ∼166 pM) of protein in sample solutions. The reported hydrogel is promising for detection of low abundance proteins while being less laborious than enzyme-linked immunosorbent assay and less affected by changes in environmental conditions than label-free biosensors.

A pin-based pyro-electrohydrodynamic jet sensor for tuning the accumulation of biomolecules down to sub-picogram level detection

The detection of low abundant biomarkers is of great interest in different clinical cases, such as the early diagnosis of Alzheimer's disease. Here we present what we call pin-based pyro-electrohydrodynamic jet (p-jet) sensor that can linearly tune the accumulation rate of biomolecules in tiny droplets. In this newly revealed pin-based configuration we demonstrate the ability to detect biomolecules down to sub-picogram concentration. Two different fluorescent probes at a varying number of accumulated droplets, up to 50, have been used to investigate the p-jet sensor. Moreover, a comparative study with a commercial piezo-driven bio-spotter has been performed. The spots of biomolecules exhibit high reproducibility by achieving a coefficient of variation <10%, both in diameter and fluorescence intensity, respectively. Remarkably, the spot intensity increases linearly with the number of accumulated droplets, thus showing the effective inherent additive functionality of this technique. Such accumulation effect allowed us to reach a limit of detection down to sub-picogram level. The p-jet sensor was applied to detect the Aβ1–42 protein, a typical Alzheimer's disease biomarker, through an immunoreaction protocol carried out on the p-jet spots. The results demonstrate the compatibility of the p-jet with immunoassay procedures, opening the route to the development of a highly sensitive device able to detect low abundant biomarkers for early diagnosis applications.

Attomolar sensitivity microRNA detection using real-time digital microarrays

MicroRNAs (miRNAs) are a family of noncoding, functional RNAs. With recent developments in molecular biology, miRNA detection has attracted significant interest, as hundreds of miRNAs and their expression levels have shown to be linked to various diseases such as infections, cardiovascular disorders and cancers. A powerful and high throughput tool for nucleic acid detection is the DNA microarray technology. However, conventional methods do not meet the demands in sensitivity and specificity, presenting significant challenges for the adaptation of miRNA detection for diagnostic applications. In this study, we developed a highly sensitive and multiplexed digital microarray using plasmonic gold nanorods as labels. For proof of concept studies, we conducted experiments with two miRNAs, miRNA-451a (miR-451) and miRNA-223-3p (miR-223). We demonstrated improvements in sensitivity in comparison to traditional end-point assays that employ capture on solid phase support, by implementing real-time tracking of the target molecules on the sensor surface. Particle tracking overcomes the sensitivity limitations for detection of low-abundance biomarkers in the presence of low-affinity but high-abundance background molecules, where endpoint assays fall short. The absolute lowest measured concentration was 100 aM. The measured detection limit being well above the blank samples, we performed theoretical calculations for an extrapolated limit of detection (LOD). The dynamic tracking improved the extrapolated LODs from femtomolar range to sim 10 attomolar (less than 1300 copies in 0.2 ml of sample) for both miRNAs and the total incubation time was decreased from 5 h to 35 min.

Highly sensitive monitoring of telomerase activity in living cells based on rapidly triggered cascade amplification reaction

Telomerase is an important potential biomarker for the study of tumor progression. Herein, we designed a cascade-amplification-reaction-based nanoprobe for intracellular telomerase detection based on the integration of rolling circle amplification (RCA) and catalytic hairpin assembly (CHA) onto MnO2 nanosheets. Firstly, MnO2 nanosheets rapidly delivered and released signal amplification units into cells, and very short telomerase extension products formed RCA circular templates and initiated the exponential RCA, producing enriched telomere sequence amplification products. Then the amplification products specifically triggered the CHA process and numerous H1/H2 complexes were formed, realizing the exponential amplification of fluorescence signals. The detection limit is as low as 1 LoVo cell for telomerase activity in cell extract. We further designed a microfluidic chip with six independent cell culture regions for in situ fluorescence imaging. Simultaneous detection of six types of cells was realized on the chip, and only 1–2 μL of cell suspension and reagents are needed. Our detection method features faster response speed and stronger fluorescence signal. Telomerase in living cells showed strong fluorescence signal within 1.5 h, and tumor cells were effectively distinguished from normal cells. Telomerase activities of different types of tumor cells and activity changes were both monitored conveniently. These results demonstrate that this method holds the potential for the sensitive detection of low abundance biomarkers in living cells, and will contribute to cancer diagnosis, cancer treatment and telomerase-related drug screening.

Catalytic hairpin self-assembly regulated chameleon silver nanoclusters for the ratiometric detection of CircRNA

CircRNA, a group of circular, closed, single-stranded non-coding RNA, is an important biomarker for cancer diagnosis and prognosis. Sensitive and selective detection of circRNA can greatly facilitate the early diagnosis of human diseases. Herein, an isothermal amplification system is proposed based on dual-catalyzed hairpin self-assembly (CHA) and chameleon DNA templated silver nanoclusters (DNA-AgNCs) for the label-free ratiometric detection of circRNA. The upstream CHA1 could be specifically triggered by the analyte to form double-stranded DNA (dsDNA) products, leading to the red fluorescence of hairpin DNA-AgNCs decreased due to the destruction of the hairpin structure. The resulting dsDNA, with a trigger sequence, could then further activate downstream CHA2 to generate another dsDNA complex, which induced the other dark AgNCs to approach the G-rich sequence, thereby causing a dramatic increase in green fluorescence. Thus, by measuring the distinct variation in the ratios of green and red fluorescence intensities, the ratiometric system could be used to sensitively detect and visually distinguish the circRNA. In addition, the two-stage signal amplification in the CHA cycle endowed the detection method with ultra-sensitive detection performance, in which the detection limits for the RNA target were honed to 1 pM. Moreover, this novel method can be used as a general strategy to analyze different types of circRNA, thus showing its great potential for the detection of low abundant biomarkers in various clinical research studies.

Sub-picomolar lateral flow antigen detection with two-wavelength imaging of composite nanoparticles

Lateral flow tests, commonly based on metal plasmonic nanoparticles, are rapid, robust, and low-cost. However, improvements in analytical sensitivity are required to allow detection of low-abundance biomarkers, for example detection of low antigen concentrations for earlier or asymptomatic diagnosis of infectious diseases. Efforts to improve sensitivity often require changes to the assay. Here, we developed optical methods to improve the sensitivity of absorption-based lateral flow tests, requiring no assay modifications to existing tests. We experimentally compared five different lock-in and subtraction-based methods, exploiting the narrow plasmonic peak of gold nanoparticles for background removal by imaging at different light wavelengths. A statistical framework and three fitting models were used to compare limits of detection, giving a 2.0–5.4-fold improvement. We then demonstrated the broad applicability of the method to an ultrasensitive assay, designing 530 nm composite nanoparticles to increase the particle volume, and therefore light absorption per particle, whilst retaining the plasmonic peak to allow background removal and without adding any assay steps. This multifaceted, modular approach gave a combined 58-fold improvement in the fundamental limit of detection using a biotin-avidin model over 50 nm gold nanoparticles with single-wavelength imaging. Applying to a sandwich assay for the detection of HIV capsid protein gave a limit of detection of 170 fM. Additionally, we developed an open-source software tool for performing the detection limit analysis used in this work.

Dual amplification enabled counting based ultrasensitive enzyme-linked immunosorbent assay

ELISA is a predominant technique in the detection of biomarkers. Notwithstanding its ubiquity and numerous advantages, its application for detection of low abundant biomarkers requires the ultrasensitivity. To bridge the gap between the need and availability, an innovative dual amplification enabled counting based ELISA was developed for ultrasensitive detection of mouse total IgG (a model biomarker). The dual amplification strategy, which is compatible with conventional plate-based ELISA, was realized through tyramide signal amplification (TSA) and alkaline phosphatase enabled formation of fluorescent precipitates. The counting of fluorescent precipitates in 25 images of each well can correlate the number of precipitates to the concentration of IgG with good linearity and lower the limit of detection of the commercial mouse IgG kit (1.56 ng/mL) to 54.5 pg/mL. Recovery tests further demonstrate the reliability of the developed method. This study opens a new avenue to improve the sensitivity of conventional plate-based ELISA.

High Resolution Continuous Elution Electrophoresis for the Evaluation of Low Abundance Serum Proteins.

The evaluation of low abundance biomarkers in the circulating low molecular weight serum proteome is an important source of information. Techniques for sample preparation to remove high abundant proteins and to enrich the low molecular weight fraction are usually required prior to novel biomarker detection. A continuous elution electrophoresis was used to separate the low molecular weight serum proteins from the high abundance serum proteins, such as albumin and immunoglobulins. Centrifugal concentration, SDS-PAGE, and total protein staining were performed to analyze eluted protein fractions. Consecutive concentrated serum protein fractions demonstrate separation at a high resolution of 1 - 2 kDa below 20 kDa. Continuous elution electrophoresis is an adequate method to eliminate high abundance proteins which interfere with the detection of low abundance biomarkers in the low molecular weight proteome and to enrich its proteins for subsequent detection and clinical evaluation.

Smartphone Enabled Point-of-Care Detection of Serum Biomarkers.

Sandwich immunoassays are the gold standard for detection of protein analytes. Here, we describe an ultrasensitive point-of-care sandwich immunoassay platform for the detection of biomarkers directly from blood or serum using a custom-built smartphone detector. Testing undiluted blood or serum is challenging due to the complexity of the matrix. Proteins nonspecifically adsorb to and cells often adhere to the assay surface, which can drastically impact the analytical sensitivity of the assay. To address this problem, our assay is built upon a "nonfouling" polymer brush "grafted from" a glass slide, which eliminates nearly all nonspecific binding and therefore increases the signal-to-noise ratio and greatly improves the analytical performance of the test. The two components required to perform a sandwich immunoassay are inkjet-printed directly onto the surface: (1) "stable" capture antibodies that remain entrapped in the brush even after exposure to a liquid sample and (2) fluorescently labeled "soluble" detection antibodies that dissolve upon exposure to a liquid sample. The polymer brush provides hydration to the antibodies, allowing them to remain stable and active over prolonged periods of time. When a liquid sample containing a biomarker of interest is dispensed onto the chip, the detection antibodies dissolve and diffuse to the stable capture spots forming a complex that sandwiches the analyte and that has a fluorescence intensity proportional to the concentration of the biomarker in solution, which can be measured using a custom-built smartphone detector. As multiple capture antibodies can be printed as discrete capture spots, the assay can be easily multiplexed without the need for multiple fluorophores. This chip and detector platform can be utilized for the point-of-care detection of low-abundance biomarkers directly from blood or serum in low-resource settings.

Distance-based quantification of miRNA-21 by the coffee-ring effect using paper devices.

Enabled by the coffee-ring effect, a paper-based signal transduce method is employed for catalytic hairpin assembly (CHA) amplification and hybridization chain reaction (HCR) to achieve miRNA quantification. Once the target miRNAs appeared, it was circularly used by CHA to initiate HCR amplification to produce a large number of G-quadruplex, which is combined with hemin to form a hemin/G-quadruplex DNAzyme. The DNAzyme catalyzes a colorimetric reaction to produce colored nanoparticles, which were converted to the end edge of the paper by evaporation-driven flow, forming a visible colored band. Higher concentration of miRNA led to more colored nanoparticles and thus a longer colored band that can simply be measured by a ruler. The results of determination of miRNA in samples demonstrate that the relative standard deviation of the proposed approach is 5.2%, highly sensitive and repeatable, with a working range 1.0 to 1000 pM and a LOD of 0.2 pM. The paper-based analytical device as a novel platform offers a new signal transduce pathway toward the detection of low-abundance biomarkers for diagnosis.Graphical abstract Schematic representation of the principle for quantification of miRNA on paper based on the coffee-ring effect.

Self-Tracking Multifunctional Nanotheranostics for Sensitive miRNA Imaging Guided Photodynamic Therapy.

A theranostic system that enables monitoring the delivery process and amplified detection of low-abundance biomarkers is significant for bioimaging and biomedicine. We here develop a graphitic carbon nitride (g-C3N4) nanosheet based self-tracking multifunctional nanotherapeutic system for sensitive miRNA guided photodynamic therapy in living cells. The g-C3N4 nanosheets are employed not only as a nanocarrier for an intracellular hybridization chain reaction for sensitive miRNA imaging but also as an excellent photosensitizer for tumor therapy. Moreover, the fluorescent g-C3N4 nanosheets allow assessing the transfection efficiency to mitigate the false negative signals. In vitro experiments showed that the proposed strategy had high signal amplification efficiency. The live cell studies revealed that the g-C3N4@H1/H2 could distinguish different miRNA expression levels from tumor to normal cells. The cell viability assay demonstrated the good photodynamic therapy efficiency of g-C3N4@H1/H2. Therefore, the developed self-tracking multifunctional nanotheranostics hold great potential for biomarker detection and imaging guided photodynamic therapy in living cells.

DNA Amplifier-Functionalized Metal-Organic Frameworks for Multiplexed Detection and Imaging of Intracellular mRNA.

DNA amplification is a useful technique for low-abundance biomarker detection and environmental monitoring because of its high signal-amplifying ability. However, intracellular application of DNA amplifiers remains challenging due to poor delivery efficiency and stability. Herein, we report an entropy-driven DNA amplifier-functionalized metal-organic framework (DNA amplifier-MOF) for the detection and imaging of multiple intracellular messenger RNAs (mRNAs). The DNA amplifier-MOF conjugate exhibits high cellular uptake, enhanced enzymatic stability, and good biocompatibility. Importantly, in the presence of phosphate ions, a surface-functionalized DNA amplifier can be released in cells with high efficiency, which facilitates the imaging of mRNA. This method is rapid and of high sensitivity/specificity, as validated in HepG2 and HL7702 cells for the imaging of TK1 and survivin mRNA, respectively. With further optimization, the strategy can become a powerful biotechnology tool for the detection of cancers at early stages and for elucidating biological processes.

Signal Amplification for Imaging Mass Cytometry.

An enzyme-catalyzed reporter deposition stain has been developed for Imaging Mass Cytometry (IMC). The reagent consists of an alkaline phosphatase substrate tethered to a tellurophene which serves as reporter group for mass cytometry. Upon phosphate hydrolysis, a quinone methide is released which covalently labels local nucleophiles. This strategy is a useful complement to heavy isotope antibody conjugates as it facilitates signal amplification for low-abundance biomarker detection. The workflow is conveniently integrated with standard IMC antibody staining to allow multiparametric antigen detection.

A facile DNA/RNA nanoflower for sensitive imaging of telomerase RNA in living cells based on “zipper lock-and-key” strategy

The sensitive imaging of telomerase RNA (TR) in living cells is crucial for improved guidance in cancer clinical diagnosis because its expression level is closely related to malignant diseases. The efficient delivery of multiple nucleic acid probes to target cells is critical for nucleic acid-based methods to successfully image low-abundance TR in living cells. While novel nanomaterials enhance delivery efficiency, uncontrolled loading and slow intracellular release remain major challenges for multiple-probe delivery. Here, we designed a facile DNA/RNA nanoflower (NF) to perform the controlled loading of multiple probes and rapid intracellular release based on the “zipper lock-and-key” strategy. First, a long RNA generated by rolling circle transcription acts as both the “smart zipper lock” and the delivery carrier to alternately lock multiple functional DNAs through DNA-RNA base pairing, and the resulting RNA/DNA hybrids self-assemble into packed NFs. The functional DNAs include the fluorescence molecular beacon H1 for TR recognition, H2 for hybrid chain reaction (HCR) and DNA-cholesterol for size control. After NF internalization by the cells, the intracellular RNase H acts as the “key” to specifically open the DNA/RNA NFs by cleaving the RNA in the DNA/RNA hybrid, releasing high amounts of H1 and H2 in a confined space and thereby facilitating the HCR amplification analysis of cytoplasmic TR. With the addition of a DNA-nuclear localization peptide component in the same NF, nuclear TR can also be sensitively detected. Compared with the regular H1/H2 mixture, the DNA/RNA NFs produced a higher-contrast fluorescence signal. This indicated that the proposed strategy allowed the side arms of H1/H2 to be sealed into the RNA sequence-programmed “zipper lock” by controlled loading, avoiding mutual nonspecific H1/H2 hybridization. In addition, due to the fast kinetics of the RNase endonuclease reaction, the loaded H1/H2 was quickly released. Furthermore, the strategy was successfully used to assay the expression levels of TR in HeLa, HepG2 and HL-7702 cells, demonstrating that this approach holds the potential for the sensitive detection of low-abundance biomarkers in living cells.

Jungle on the Electrode: A Target-Induced Enzyme-Free and Label-Free Biosensor.

With the development of clinical medicine-related technologies, the detection of cancer biomarkers has become an essential method for cancer diagnosis. DNA self-assembly is a valuable approach to monitor low-abundance miRNA. Herein, we report a novel DNA jungle biosensor for target-induced enzyme-free and label-free ultrasensitive detection of miRNA-21 in biological samples. The method consists of two parts. The target catalyzes the assembly of the hairpin to form the backbone of the DNA jungle, and the auxiliary probes hybridize to form branches freely and undirectedly. The DNA jungle can be self-assembled seeded from a single target initiator, affording the potential for screening a single target molecule. Vitro assays show that the DNA jungle offers very high amplification efficiency and specificity, with a direct detection limit of 1 aM. This DNA jungle strategy can provide a useful platform for low-abundance biomarker detection for cell biology and diagnostics.

Catalytic Self-Assembly of Quantum-Dot-Based MicroRNA Nanosensor Directed by Toehold-Mediated Strand Displacement Cascade.

Semiconductor quantum dots (QDs) are highly attractive nanomaterials with wide biomedical applications owing to their unique photophysical properties. However, the adaptation of the nonenzymatic QD nanosensor assembly to sense low-abundance targets remains a great challenge. Herein, taking advantage of the dynamic DNA nanotechnology and single-molecule fluorescence detection, we demonstrate the catalytic self-assembly of a QD-based microRNA nanosensor directed by toehold-mediated strand displacement cascade for the simple and sensitive detection of microRNA at femtomolar concentration without the requirement of any enzymes. Moreover, this QD nanosensor is capable of detecting circulating microRNA in clinical serum samples and even imaging microRNA in living cells. This work may extend the use of an enzyme-free QD nanosensor assembly for low-abundance biomarker detection and offer a novel platform for fundamental biomedical research and clinical applications.

Branched Polyethylenimine-Modified Upconversion Nanohybrid-Mediated Photoelectrochemical Immunoassay with Synergistic Effect of Dual-Purpose Copper Ions

This work developed a near-infrared (near-IR) light-activated non-enzymatic signal-off photoelectrochemical (PEC) immunoassay for the ultrasensitive detection of α-fetoprotein (AFP) on the basis of branched polyethylenimine (BPEI)-modified upconversion nanoparticle (UCNP)@CdTe quantum dot (QD) nanostructures by coupling with the synergistic effect of dual-purpose copper ions. Emission light originated from NaYF4:Yb,Er UCNP was directly utilized through the electrostatic bonding of CdTe QDs to excite the separation of electron-hole pairs, resulting in the generation of obvious photocurrent under a 980 nm laser. By using polyclonal antibody-labeled cupric oxide nanoparticle as the secondary antibody, the nanolabel was introduced into the monoclonal anti-AFP antibody-modified microplates in the presence of target AFP. After treatment with acid, the as-released copper ion decreased the photocurrent through the synergistic effect with two issues: one was initially to form coordination with BPEI on the surface of UCNP, and then the near-IR excitation light and upconversion luminescence were attenuated due to the internal filter effect; another was to snatch the electrons flowing from the valence band of CdTe QD as the exciton trapping sites. Under optimal conditions, the dual-purpose Cu2+-activated signal-off PEC immunosensing platform exhibited a dynamic linear range from 10 pg mL-1 to 50 ng mL-1, accompanying the decreasing photocurrent with the increment of AFP concentration at an experimental detection limit of 1.2 pg mL-1. Importantly, good accuracy was achieved by this method in comparison with the results with human AFP ELISA kit for analysis of human serum samples. This dual-purpose Cu2+-activated PEC immunoassay brings a promising, enzyme-free and innovative thinking for the detection of low-abundance biomarkers.