Simultaneous detection of multiple microRNAs based on fluorescence resonance energy transfer under a single excitation wavelength.

MicroRNAs (miRNAs) play a key role in physiological processes, and their dysregulation is closely related to various human diseases. Simultaneous detection of multiple miRNAs is pivotal to cancer diagnosis at an early stage. However, most multicomponent analyses generally involve multiple excitation wavelengths, which are complicated and often challenging to simultaneously acquire multiple detection signals. In this study, a convenient and sensitive sensor was developed to simultaneously detection of multiple miRNAs under a single excitation wavelength through the fluorescence resonance energy transfer between the carbon dots (CDs)/quantum dots (QDs) and graphene oxide (GO). A hybridization chain reaction (HCR) was triggered by miRNA-141 and miRNA-21, resulting in the high sensitivity with a limit of detection (LOD) of 50 pM (3σ/k) for miRNA-141 and 60 pM (3σ/k) for miRNA-21. This simultaneous assay also showed excellent specificity discrimination against the mismatch. Furthermore, our proposed method successfully detected miRNA-21 and miRNA-141 in human serum samples at a same time, indicating its diagnostic potential in a clinical setting.

DNA Nanospheres Assisted Spatial Confinement Signal Amplification for MicroRNA Imaging in Live Cancer Cells.

DNA assemblies are commonly used in biosensing, particularly for the detection and imaging of microRNAs (miRNAs), which are biomarkers associated with tumor progression. However, the difficulty lies in the exploration of high-sensitivity analytical techniques for miRNA due to its limited presence in living cells. In this study, we introduced a DNA nanosphere (DS) enhanced catalytic hairpin assembly (CHA) system for the detection and imaging of intracellular miR-21. The single-stranded DNA with four palindromic portions and extending sequences at the terminal was annealed for assembling DS, which avoided the complex sequence design and high cost of long DNA strands. Benefiting from the multiple modification sites of DS, functional hairpins H1 (modified with Cy3 and BHQ2) and H2 were grafted onto the surface of DS for assembling DS-H1-H2 using a hybridization reaction. The DS-H1-H2 system utilized spatial confinement and the CHA reaction to amplify fluorescence signals of Cy3. This enabled highly sensitive and rapid detection of miR-21 in the range from 0.05 to 3.5 nM. The system achieved a limit of determination (LOD) of 2.0 pM, which was 56 times lower than that of the control CHA circuit with freedom hairpins. Additionally, the sensitivity was improved by 8 times. Moreover, DS-H1-H2 also showed an excellent imaging capability for endogenous miR-21 in tumor cells. This was due to enhanced cell internalization efficiency, accelerated reaction kinetics, and improved biostability. The imaging strategy was shown to effectively monitor the dynamic content of miR-21 in live cancer cells and differentiate various cells. In general, the simple nanostructure DS not only enhanced the detection and imaging capability of the conventional probe but also could be easily integrated with the reported DNA-free probe, indicating a wide range of potential applications.

Fe-doped carbon dots: a novel fluorescent nanoprobe for cellular hypochlorous acid imaging.

Real-time monitoring of hypochlorous acid (HClO) in biological systems is of great significance for exploring and regulating its pathological functions because abnormal production of HClO is closely related with many diseases, such as atherosclerosis, rheumatoid arthritis, and cancer. Herein, we developed a reliable fluorescent Fe-doped carbon dots (Fe-CDs) for the sensitive and selective detection of biological HClO using ferrocenecarboxylic acid and m-phenylenediamine as precursors through a one-step solvothermal procedure. The Fe-CDs exhibited excellent a wide HClO detection range from 20 nmol/L to 1000 nmol/L with corresponding limits of detection at 7.8 nmol/L. The sensing mechanism is based on the chemical oxidation of the hydroxyl groups on the surface of Fe-CDs by HClO. In addition, Fe-CDs also displayed high photoluminescence yield, excitation-independence emission, as well as good biocompatibility, enabling the successful imaging of endogenous and exogenous HClO in HeLa cells. These results revealed that Fe-CDs holds great promise as a robust fluorescent probe for investigating HClO-mediated biological events.

Rational design of genotyping nanodevice for HPV subtype distinction.

There are more than 200 subtypes of human papillomavirus (HPV), and high-risk HPVs are a leading cause of cervical cancer. Identifying the genotypes of HPV is significant for clinical diagnosis and cancer control. Herein, we used programmable and modified DNA as the backbone to construct fluorescent genotyping nanodevice for HPV subtype distinction. In our strategy, the dye-labeled single-stranded recognize-DNA (R-DNA) was hybridized with Black Hole Quencher (BHQ) labeled single-stranded link-DNA (L-DNA) to form three functionalized DNA (RL-DNA). Through the extension of polycytosine (poly-C) in L-DNA, three RL-DNAs can be more firmly adsorbed on graphene oxide to construct reliable genotyping nanodevice. The genotyping nanodevice had low background noise since the dual energy transfer, including Förster resonance energy transfer (FRET) from dye to BHQ and the resonance energy transfer (RET) from dye to graphene oxide. Meanwhile, the programmability of DNA allows the proposed strategy to simultaneously and selectively distinguish several HPV subtypes in solution using DNA labeled with different dyes. To demonstrate clinical potential, we show multiplexed assay of HPV subtypes in cervical scrapes, and it has been successfully applied in HPV-DNA analysis in cervical scrapes samples. The genotyping nanodevice could be developed for simultaneous and multiplex analysis of several oligonucleotides in a homogeneous solution by adjusting the recognition sequence, demonstrating its potential application in the rapid screening of multiple biomarkers.

Simultaneous Response of Mitochondrial MicroRNAs to Identify Cell Apoptosis with Multiple Responsive Intelligent DNA Biocomputing Nanodevices.

Challenges remained in precisely real-time monitoring of apoptotic molecular events at the subcellular level. Herein, we developed a new type of intelligent DNA biocomputing nanodevices (iDBNs) that responded to mitochondrial microRNA-21 (miR-21) and microRNA-10b (miR-10b) simultaneously which were produced during cell apoptosis. By hybridizing two hairpins (H1 and H2) onto DNA nanospheres (DNSs) that had been previously modified with mitochondria-targeted triphenylphosphine (TPP) motifs, iDBNs were assembled in which two localized catalytic hairpins self-assembly (CHA) reactions occurred upon the co-stimulation of mitochondrial miR-21 and miR-10b to perform AND logic operations, outputting fluorescence resonance energy transfer (FRET) signals for sensitive intracellular imaging during cell apoptosis. Owing to the spatial confinement effects of DNSs, it was discovered that iDBNs had a high efficiency and speed of logic operations by high local concentrations of H1 and H2, making the simultaneous real-time responses of mitochondrial miR-21 and miR-10b reliable and sensitive during cell apoptosis. These results demonstrated that iDBNs were simultaneously responsive to multiple biomarkers, which greatly improved the detection accuracy to identify the cell apoptosis, demonstrating that iDBNs are highly effective and reliable for the diagnosis of major disease and screening of anticancer drugs.

In Situ Activation of the Receptor Aggregation for Cell Apoptosis by an AI-Au Intelligent Nanomachine via Tumor Extracellular Acidity.

Polyvalent ligand-induced cell receptor aggregation is closely related to cell behavior regulation. At present, most of the means to induce receptor aggregation rely on external stimuli such as light, heat, and magnetic fields, which may cause side effects to normal cells. How to achieve receptor aggregation on the cancer cell surface to achieve cell apoptosis selectively is still a challenge. Therefore, by taking advantage of the unique property of cancer cells' slightly acidic microenvironment, an easy-to-use apoptosis-inducing mode for the in situ activation of cell surface nucleolin clustering has been developed, which not only opened a new channel for nucleolin receptor aggregation to regulate cell function and further development but also avoided damage to normal cells, providing a new strategy for tumor treatment. Dual functional ssDNA (AS1411 aptamer and pH-responsive I-strand sequence) was modified on the surface of gold nanoparticles (AuNPs) to fabricate AI-Au intelligent nanomachines. Then, the specific binding on cancer cells and aggregation of the nucleolin receptors can be achieved via the formation of an i-Motif structure among adjacent AuNPs under the acidic microenvironment. The result showed that AI-Au nanomachines mediated nucleolin cross-linking on the cell surface, resulting in a cytotoxic effect of approximately 60%. Experiments such as calcein-AM/PI staining, nuclear dye staining, and flow cytometry demonstrated that cell apoptosis became more evident with the increase of acidity under the cell surface microenvironment. Immunofluorescence imaging further confirmed the Cyt-c/caspase-3 apoptosis pathway induced by AI-Au nanomachines. The proposed strategy used for specific cancer cell apoptosis by the in situ activation of tumor cell membrane receptor aggregation is inexpensive and simple to use, which not only provides a new means of nucleolin receptor aggregation for regulating cell function but also offers a new strategy for tumor treatment with reduced side effect to normal cells. This work is significant for comprehending the ligand-induced receptor aggregation process and can lead to the development of a promising anticancer drug.

DNA-scaffolded multivalent vaccine against SARS-CoV-2.

Short peptides are poor immunogens. One way to increase their immune responses is by arraying immunogens in multivalency. Simple and efficient scaffolds for spatial controlling the inter-antigen distance and enhancing immune activation are required. Here, we report a molecular vaccine design principle that maximally drives potent SARS-CoV-2 RBD subunit vaccine on DNA duplex to induce robust and efficacious immune responses in vivo. We expect that the DNA-peptide epitope platform represents a facile and generalizable strategy to enhance the immune response. STATEMENT OF SIGNIFICANCE: DNA scaffolds offer a biocompatible and convenient platform for arraying immunogens in multivalency antigenic peptides, and spatially control the inter-antigen distance. This can effectively enhance immune response. Peptide (instead of entire protein) vaccines are highly attractive. However, short peptides are poor immunogens. Our DNA scaffolded multivalent peptide immunogen system induced robust and efficacious immune response in vivo as demonstrated by the antigenic peptide against SARS-CoV-2. The present strategy could be readily generalized and adapted to prepare multivalent vaccines against other viruses or disease. Particularly, the different antigens could be integrated into one single vaccine and lead to super-vaccines that can protect the host from multiple different viruses or multiple variants of the same virus.

Two-Dimensional Analysis Method for Highly Sensitive Detection of Dual MicroRNAs in Breast Cancer Cells.

Multiple biomarker detection is crucial for early clinical diagnosis, and it is significant to achieve the simultaneous detection of multiple biomarkers with the same nanomaterial. In this work, the hairpin DNA strands were selectively modified on the surface of gold nanorods (AuNRs) to construct two kinds of nanoprobes by rational design. When in the presence of dual microRNAs, AuNRs were assembled to form end-to-end (ETE) and side-by-side (SBS) dimers. Compared with a single AuNR, the dark-field scattering intensity and red color percentage variation of dimers were extremely distinguished, which could be developed for dual microRNA detection by combining the red color percentage and scattering intensity with the data processing method of principal component analysis to construct a two-dimensional analysis method. Especially, the fraction of AuNR dimers presented a linear relationship with the amount of microRNAs. Based on this, microRNA-21 and microRNA Let-7a in breast cancer cells were detected with the detection limits of 1.72 and 0.53 fM, respectively. This method not only achieved the sensitive detection of dual microRNAs in human serum but also realized the high-resolution intracellular imaging, which developed a new way for the oriented assembly of nanomaterials and biological detection in living cells.

Dark-Field Imaging Monitoring of Adenosine Triphosphate in Live Cells by Au NBPs@ZIF-8 Nanoprobes.

Monitoring the fluctuation of adenosine triphosphate (ATP) level in living cells could promote the understanding of metabolic pathways and cell biology. Here, we proposed a highly sensitive, selective, and biocompatible nanoprobe with core-shell structure, namely Au NBPs@ZIF-8 composed by gold nanobipyramids (Au NBPs) and zeolitic imidazolate framework-8 (ZIF-8), for monitoring intracellular ATP level fluctuation in living cells. Because the coordination between ATP and Zn2+ (the metal node of ZIF-8) was much stronger than that between 2-methylimidazole and Zn2+, which caused the decomposition of the ZIF-8 shell and the exposure of Au NBPs in the presence of ATP, it led to the change of the localized surface plasmon resonance scattering properties of nanoprobes under dark-field microscopy. Tricolor (RGB) analysis showed that R/G value had a good linear relationship with the ATP concentrations in the range of 10 µM to 4 mM (R2 = 0.999) with a detection limit of 5.28 µM. This ATP sensing platform also exhibited excellent selectivity in complex intracellular interfering substances. Besides, we realized intracellular ATP real-time imaging in HeLa cells and observed the ATP level fluctuation under dark-field microscopy. The method mentioned here could be further applied for delivery of therapeutics for biomedical applications.

Precision medicine-guided co-delivery of ASPN siRNA and oxaliplatin by nanoparticles to overcome chemoresistance of colorectal cancer.

The development of chemoresistance is a major hurdle for the treatment of colorectal cancer (CRC), which contributes remarkably to the poor clinical prognosis. Nanodrug delivery systems show great potential in overcoming chemoresistance, but limited by the lack of identification of chemoresistance targets from cancer patients. In the present study, we enrolled chemotherapy-resistant or sensitive CRC patients and used the next-generation RNA sequencing to reveal that Asporin (ASPN) is highly expressed in tumor tissues from oxaliplatin (OXA)-resistant patients and closely correlated with a poor prognosis of CRC. Downregulation of ASPN reversed OXA resistance and promoted cell apoptosis both in vitro and in vivo. To overcome ASPN-mediated OXA resistance, we constructed a nanoparticle-based co-delivery system (denoted as PPO-siASPN) for simultaneous delivery of OXA and siRNA targeting ASPN (siASPN). PPO-siASPN not only facilitated the intracellular delivery of OXA through the enhanced cellular uptake, but effectively suppressed ASPN expression for synergistic antitumor activity in vitro and in vivo. In the more clinically relevant patient-derived xenograft (PDX) mouse model, systemic administration of PPO-siASPN achieved a remarkable therapeutic effect. This study uncovered the critical role of ASPN in causing OXA resistance in CRC patients and suggests a promising nanoformulation that may be more effective than current standard-of-care medications.

DNA Logic Nanodevices for the Sequential Imaging of Cancer Markers through Localized Catalytic Hairpin Assembly Reaction.

Monitoring tumor biomarkers is crucial for cancer diagnosis, progression monitoring, and treatment. However, identifying single or multiple biomarkers with the same spatial locations can cause false-positive feedback. Herein, we integrated the DNA tetrahedron (DT) structures with logic-responsive and signal amplifying capability to construct transmembrane DNA logic nanodevices (TDLNs) for the in situ sequential imaging of transmembrane glycoprotein mucin 1 (MUC1) and cytoplasmic microRNA-21 (miR-21) to cell identifications. The TDLNs were developed by encoding two metastable hairpin DNAs (namely, H1 and H2) in a DT scaffold, in which the triggering toeholds of H1 for miR-21 were sealed by the MUC1-specific aptamer (MUC1-apt). The TDLNs not only had the function of signal amplification owing to the localized catalytic hairpin assembly (CHA) reaction through spatial constraints effect of DT structures but also performed an AND logic operation to output a green Cy3 signal in MCF-7 cells, where MUC1 protein and miR-21 were simultaneously expressed. These results showed that the newly developed TDLNs have better molecular targeting and recognition ability so as to be easily identify cell types and diagnose cancer early.

The restructure of Au@Ag nanorods for cell imaging with dark-field microscope.

The facile and noninjurious image of cells with high resolution and low toxicity is essential since imaging can offer rich and direct information and insights into metabolic activities, clinical diagnosis, drug delivery and cancer therapy. In this contribution, a smart imaging probe was employed as a contrast agent for dark-field cell imaging. Au core/Ag shell nanorods (Au@Ag NRs) that characterized by X-ray diffraction and X-ray photoelectron spectroscopy, formed Au@Ag@AgI NRs when exposed to iodine, which greatly enhanced the light scattering of nanorods. Herein, the silver shell acted as the response element for iodine as well as the protective agent for Au core. When conjugated with folate, the nanorods can be used to image human cervical cancer cells (HeLa cells) under a dark-field microscope. Nanorods were demonstrated with excellent tumor cellular uptake ability without obvious cytotoxicity, making them ideal candidates in biosensing and bioimaging applications.

A high-integrated DNA biocomputing platform for MicroRNA sensing in living cells.

DNA logic computing has captured increasing interest due to its ability to assemble programmable DNA computing elements for disease diagnosis, gene regulation, and targeted therapy. In this work, we developed an aptamer-equipped high-integrated DNA biocomputing platform (HIDBP-A) with a dual-recognition function that enabled cancer cell targeting. Dual microRNAs were the input signals and can perform AND logic operations. Compared to the free DNA biocomputing platform (FDBP), the integration of all computing elements into the same DNA tetrahedron greatly improved logic computing speed and efficiency owing to the confinement effect reflected by the high local concentration of computing elements. As a proof of concept, the utilization of microRNA as the input signal was beneficial for improving the scalability and flexibility of the sequence design of the logic nano-platform. Given that the different microRNAs were over-expressed in cancer cells, this new HIDBP-A has great promise in accurate diagnosis and logic-controlled disease treatment.

Simultaneous Imaging of Dual microRNAs in Cancer Cells through Catalytic Hairpin Assembly on a DNA Tetrahedron.

Accurate detection and imaging of tumor-related microRNA (miRNA) in living cells hold great promise for early cancer diagnosis and prognosis. One of the challenges is to develop methods that enable the identification of multiple miRNAs simultaneously to further improve the detection accuracy. Herein, a simultaneous detection and imaging method of two miRNAs was established by using a programmable designed DNA tetrahedron nanostructure (DTN) probe that includes a nucleolin aptamer (AS1411), two miRNA capture strands, and two pairs of metastable catalytic hairpins at different vertexes. The DTN probe exhibited enhanced tumor cell recognition ability, excellent stability and biocompatibility, and fast miRNA recognition and reaction kinetics. It was found that the DTN probe could specifically enter tumor cells, in which the capture strand could hybridize with miRNAs and initiate the catalytic hairpin assembly (CHA) only when the overexpressed miR-21 and miR-155 existed simultaneously, resulting in a distinct fluorescence resonance energy transfer signal and demonstrating the feasibility of this method for tumor diagnosis.

Plasmonic biosensor for the highly sensitive detection of microRNA-21 via the chemical etching of gold nanorods under a dark-field microscope.

MicroRNAs involved in tumor-related tissues at abnormal expression level present tremendous potential in the early diagnosis of cancers. However, their intrinsic shortcomings, for instance, low abundance and high sequence homology, make it challengeable to quantify them with high sensitivity and selectivity. Herein, a highly sensitive platform with great specificity was developed for microRNA-21 based on the produced-I2 triggered chemical etching of gold nanorods to a smaller size, resulting in a significant blue shift and a great intensity decrease in the localized surface plasmon resonance (LSPR) scattering. The synergism of strand displacement and enzymatic reaction enabled the proposed strategy with a high sensitivity and selectivity toward microRNA-21 in a dynamic range from 0.1 to 10,000 pM and a low limit of detection of 71.22 fM (3σ/k) by dark-field microscope. Additionally, the remarkable discrimination of single nucleotide difference suggested the superior selectivity towards microRNA-21, which presented a satisfactory recovery in human serum samples. The proposed plasmon platform could also serve as a universal and sensitive detection of cancer biomarkers, presenting the amusing application prospects in the early diagnosis of various cancers by adapting the corresponding nucleic acid sequences.

Catalytic hairpin assembled polymeric tetrahedral DNA frameworks for MicroRNA imaging in live cells.

Dynamic DNA nanodevices-based assembly is currently well developed for a broad range of analytical applications. However, some problems persist, such as false positives, nuclease digestions, and exclusive interferences with single signal in complex cellular environment. Herein, we have established a method for imaging cellular miR-155, where it induced assembly of two tetrahedral DNA frameworks (TDFs), TDF-1 and TDF-2, both of which had four fluorescence modified hairpins (Cy3 for TDF-1 and Cy5 for TDF-2, respectively) at each angle, into polymeric tetrahedral DNA frameworks (PTDFs). The formation of PTDFs was greatly dependent on miR-155 overexpressed in breast cancer cells since miR-155 drove catalytic hairpin assembly (CHA) reaction by opening the hairpins at the vertices of TDF-1 to hybridize with TDF-2. Upon the completion of hybridization, the miR-155 was released, starting the next cycle of the CHA reaction. Measurements of atomic force microscopy (AFM) and Förster resonance energy transfer (FRET) showed that the formation of PTDFs occurred owing to the multivalent assembly of TDF-1 and TDF-2. By utilizing the formation of PTDFs, miR-155 was detected in a linear range from 0.5 nM to 30 nM with a 0.35 nM limit of determination, enabling the successful imaging of endogenous miR-155 in live cells through the FRET signal from Cy3 to Cy5. These studies demonstrated that this method significantly strengthened the resistance nuclease to digestion and stable ability with exclusive interference.

DNA Logic Nanodevices for Real-Time Monitoring of ATP in Lysosomes.

DNA logic nanodevices have prospects in molecular recognitions but still face challenges in achieving DNA computation-controlled regulation in specific compartments of living cells. By incorporating the i-motif sequence and ATP aptamers into a Y-shaped DNA (Y-DNA) structure, and applying gold nanoparticles (AuNPs) as the transporting carrier, herein we present a new type of DNA logic nanodevices to monitor the ATP levels in lysosomes of living cells. Triple energy transfers including dual fluorescent resonance energy transfers (FRETs) and a nanometal surface energy transfer (NSET) occurred in the DNA logic nanodevices. It was identified that the proposed nanodevices perform an AND logic operation to output FRET signals only when an endogenous proton and ATP simultaneously exist in the cellular microenvironment. Owing to the use of the i-motif sequence, the nanodevices have lysosome-recognizing capacity without causing alkalization of the acidic organelle, making DNA computation-controlled regulation at the level of cellular organelles achievable. These DNA logic nanodevices show high application prospects in lysosome-related cellular function and disease treatment.

Transformable Helical Self-Assembly for Cancerous Golgi Apparatus Disruption.

Golgi apparatus is a major subcellular organelle responsible for drug resistance. Golgi apparatus-targeted nanomechanical disruption provides an attractive approach for killing cancer cells by multimodal mechanism and avoiding drug resistance. Inspired by the poisonous twisted fibrils in Alzheimer's brain tissue and enhanced rigidity of helical structure in nature, we designed transformable peptide C6RVRRF4KY that can self-assemble into nontoxic nanoparticles in aqueous medium but transformed into left-handed helical fibrils (L-HFs) after targeting and furin cleavage in the Golgi apparatus of cancer cells. The L-HFs can mechanically disrupt the Golgi apparatus membrane, resulting in inhibition of cytokine secretion, collapse of the cellular structure, and eventually death of cancer cells. Repeated stimulation of the cancers by the precursors causes no acquired drug resistance, showing that mechanical disruption of subcellular organelle is an excellent strategy for cancer therapy without drug resistance. This nanomechanical disruption concept should also be applicable to multidrug-resistant bacteria and viruses.

Aggregation-Enhanced Energy Transfer for Mitochondria-Targeted ATP Ratiometric Imaging in Living Cells.

Förster resonance energy transfer (FRET) from fluorescent nanoparticles to fluorescent dyes is an attractive approach for bioanalysis in living cells. However, the luminescence of the nanoparticle donor/acceptor has not been effectively used to produce highly efficient FRET because the distance between the energy donor and energy acceptor is often larger than the effective FRET radius (about 10 nm) and the uncontrolled rotational and translational diffusion of luminophores. Here, we develop an aggregation-enhanced energy transfer strategy that can overcome the impedance for effective energy transfer. The functional nanoprobes, named TPP-CDs-FITC, are carbon dots (CDs) functionalized with triphenylphosphine (TPP) and ∼117 fluorescein 5-isothiocyanate (FITC) on the surface. In dispersed solution, the 3.8 nm TPP-CDs-FITC show weak FRET efficiency (15.4%). After TPP-instructed mitochondrial targeting, enhanced FRET efficiency (53.2%) is induced due to the aggregation of TPP-CDs-FITC selectively triggered by adenosine triphosphate (ATP) in the mitochondria. The enhanced FRET efficiency can be attributed to the joint effect of the augment of numbers of FITC acceptors within 10 nm from dispersed 117 to aggregated 5499 and the restricted rotational and translational motions of TPP-CDs donors and FITC acceptors. Ultimately, we successfully observe the fluctuations of ATP levels in the mitochondria using the aggregation-enhanced energy transfer strategy of the TPP-CDs-FITC nanodevice.

Cu vacancies enhanced photoelectrochemical activity of metal-organic gel-derived CuO for the detection of l-cysteine.

Defect engineering in the photoelectrochemical (PEC) process of photoelectrodes has been extensively studied. But insufficient attention has been received about the impact of metal vacancies (VM) in PEC process. Herein, the influence of Cu vacancies (VCu) on PEC performance of copper oxide (CuO) derived from Cu-based metal-organic gel (Cu-MOG) precursor was reported. It can be found that the presence of more VCu can improve the PEC activity of CuO photocathode by facilitating the charge separation and transfer. Moreover, the as-prepared CuO was presented as a new PEC sensor to detect l-cysteine (L-Cys) on the basis of the excellent PEC performance, which showed high sensitivity and selectivity. Good linear response of L-Cys within the range of 0.1-6 µM was performed with a detection limit of 0.04 µM. This work not only provides insights into the role of VM in the PEC process of photocathodes, but also proved the high potential applicability of CuO as a PEC device for biomolecule detection.