Assembly of Dye Molecules in Covalent Organic Frameworks for Enhanced Colorimetric Biosensing.

Colorimetric assays have been extensively investigated for biosensing applications due to their advantages of visual recognizability, ease of use, and low cost. However, advancing their development is a great challenge due to the inherent limitations of colorimetric dyes. Herein, we report a strategy to assemble dyes in covalent organic frameworks (COFs) to effectively reinforce the applicability of pH-responsive dyes in colorimetric bioassays. Experimental results reveal that three-dimensional COFs can promote the assembly of dyes through hydrogen bonding, resulting in the formation of a dye-supermolecule@COF assembly. Consequently, when sensitized at increased pH levels (e.g., hydroxyl ions), disruption of hydrogen bonds may trigger a rapid transition from their insoluble fixed state within the COFs into soluble, visibly detectable dye anions. This process can also be facilitated by increased hydrophilicity and elevated electrostatic repulsion between the dye anions and COFs, leading to the substantial release of chromogenic dye anions from the COF pores into the solution, thereby amplifying the colorimetric signal output. Therefore, by employing various synthesized dye-supermolecule@COFs as signal tags, we developed a colorimetric bioassay capable of accurately identifying breast cancer cell subtypes. This study not only highlights the effectiveness of dye-supermolecule@COFs in enhancing colorimetric biosensing but also underscores the potential of employing the COF-mediated dye assembly strategy for colorimetric assays.

Colorimetric detection of furin based on enhanced catalytic activity of G-quadruplex/hemin DNAzyme.

BACKGROUND: Rapid and sensitive colorimetric detection methods are crucial for diseases diagnosis, particularly those involving proteases like furin, which are implicated in various conditions, including cancer. Traditional detection methods for furin suffer from limitations in sensitivity and practicality for on-site detection, motivating the development of novel detection strategies. Therefore, developing a simple, enzyme-free, and rapid colorimetric analysis method with high sensitivity for furin detection is imperative. RESULTS: Herein, we have proposed a colorimetric method in this work for the first time to detect furin, leveraging the assembly of G-quadruplex/hemin DNAzyme with enhanced catalytic activity. Specifically, a peptide-DNA conjugate (PDC) comprising a furin-recognition peptide and flanking DNA sequences for signal amplification is designed to facilitate the DNAzyme assembly. Upon furin treatment, PDC cleavage triggers a cyclic catalytic hairpin assembly reaction to form the complementary double-stranded structures by hairpin 1 (HP1) and hairpin 2 (HP2), bringing the G-quadruplex sequence in HP1 closer to hemin on HP2. Moreover, the resulting G-quadruplex/hemin DNAzymes exhibit robust peroxidase-like activity, enabling the catalysis of the colorimetric reaction of ABTS2- for furin detection. Our method demonstrates high sensitivity, rapid response, and compatibility with complex sample matrices, achieving a detection limit as low as 1.1 pM. SIGNIFICANCE: The DNAzyme reported in this work exhibits robust catalytic activity, enabling high sensitivity and good efficiency for the detection. By eliminating the requirement for exogenous enzymes, our approach enables visual furin detection without expensive instrumentation and reagents, promising significant utility in biomedical and clinical diagnostic applications. Given the various design of peptide sequence and the programmability of DNA, it can be readily applied to analyzing other useful tumor biomarkers.

Simplified Electrochemical Approach for End-Point Yet Quantitative Detection of Nucleic Acids in Resource-Limited Settings.

Nucleic acid detection plays a crucial role in various aspects of health care, necessitating accessible and reliable quantification methods, especially in resource-limited settings. This work presents a simplified electrochemical approach for end-point yet quantitative nucleic acid detection. By elevating the concentration of redox species and choosing potential as the signals, we achieved enhanced signal robustness, even in the presence of interfering substances. Leveraging this robustness, we accurately measured pH-induced redox potential changes in methylene blue solution for end-point nucleic acid detection after loop-mediated isothermal amplification (LAMP). Our method demonstrated quantitative detection of the SARS-CoV-2 N gene and human ATCB gene and successful discrimination of the human BRAF V600E mutation, comparable in sensitivity to commercial kits. The developed user-friendly electrochemical method offers a simplified and reliable approach for end-point yet quantitative detection of nucleic acids, potentially expanding the benefits of nucleic acid testing in resource-limited settings.

An electrochemical biosensor based on mild reduction-activated CRISPR/Cas12a system for sensitive detection of circulating tumor cells.

Circulating tumor cell (CTC) has been a valuable biomarker for the diagnosis of breast cancer, while folate receptor is a kind of cell surface receptor glycoprotein which is overexpressed in breast cancer. In this work, we have designed and fabricated an electrochemical biosensor for sensitive detection of folate receptor-positive CTCs based on mild reduction assisted CRISPR/Cas system. Specifically, folate functionalized magnetic beads are firstly prepared to capture CTCs owing to the strong affinity between folate and the folate receptors on the surface of cells. Then, the cell membranes are treated by mild reduction so as to expose a large number of free sulfhydryl groups, which can be coupled with maleimide-DNA to introduce the signal amplified CRISPR/Cas12a system. After the trans-cleavage activity of CRISPR/Cas12a is activated, the long chain DNA modified with electroactive molecules methylene blue can be randomly cleaved into short DNA fragments, which are then captured on the graphite electrode through the host-guest recognition with cucurbit [7]uril, generating highly amplified electrochemical signal corresponding to the number of CTCs. The electrochemical biosensor not only demonstrates the sensitivity with a low detection limit of 2 cells/mL, but also highlights its excellent selectivity and stability in complex environment. Therefore, our biosensor may provide an alternative tool for the analysis of CTCs.

Target-Triggered Aggregation of Modified E. coli for Diagnosis of Ovarian Cancer.

Bacteria inherently possess the capability of quorum sensing in response to the environment. In this work, we have proposed a strategy to confer bacteria with the ability to recognize targets with quorum-sensing behavior. Meanwhile, we have successfully achieved artificial control over the target-triggered aggregation of Escherichia coli (E. coli) by modifying the bacteria surface in a new way. Furthermore, by making use of green fluorescent protein (GFP) expressed by E. coli as the output signal, the aggregation of modified E. coli can be observed with the naked eye. Therefore, via the detection of the target, MUC1, an ovarian cancer biomarker, a simple and conveniently operated method to diagnose ovarian cancer is developed in this work. Experimental results show that the developed low-background and enzyme-free amplification method enables the highly sensitive detection of MUC1, achieving a remarkable limit of detection (LOD) of 5.47 fM and a linear detection range spanning from 1 pM to 50 nM and 50 nM to 100 nM, respectively. Clinical samples from healthy donors and patients can give distant assay results, showing great potential for clinical applications of this method.

DNA-Peptide Interaction-Modulated Charge Reversal in Biomimetic Nanochannels for Simple and Efficient Detection of Histone Deacetylases.

Protein acetylation, a fundamental post-translational modification, plays a critical role in the regulation of gene expression and cellular processes. Monitoring histone deacetylases (HDACs) is important for understanding epigenetic dynamics and advancing the early diagnosis of malignancies. Here, we leverage the dynamic characteristics of DNA-peptide interactions in biomimetic nanochannels to develop a HDAC detection method. In specific, the catalysis of peptide deacetylation by HDACs triggers alterations in the charge states of the nanochannel surface to accommodate DNA molecules. Then, the interaction between DNA and peptides shifts the nanochannel surface charge from positive to negative, leading to a reversal of the ion current rectification (ICR). By calculation of the ICR ratio, quantitative detection of HDACs can be efficiently achieved using the nanochannel-based method in an enzyme-free and label-free manner. Our experimental results demonstrate that HDACs can be detected by using this method within a concentration range of 0.5-500 nM. The innate simplicity and efficiency of this strategy may render it a valuable tool for advancing both fundamental research and clinical applications in the realm of epigenetics and personalized medicine.

Multiplex Assay of Cytokines with Tunable Detection Ranges for the Precise Diagnosis of Breast Cancer.

Precise profiling of the cytokine panel consisting of different levels of cytokines can provide personalized information about several diseases at certain stages. In this study, we have designed and fabricated an "all-in-one" diagnostic tool kit to bioassay multiple inflammatory cytokines ranging from picograms per milliliter to µg/mL in a small cytokine panel. Taking advantage of the kit fabricated by the DNA-encoded assembly of nanocatalysts in dynamic regulation and signal amplification, we have demonstrated the multiplex, visual, and quantitative detection of C-reactive protein (CRP), procalcitonin (PCT), and interleukin-6 (IL-6) with limits of detection of 1.6 ng/mL (61.54 pM), 20 pg/mL (1.57 pM), and 4 pg/mL (0.19 pM), respectively. This diagnostic tool kit can work well with commercial kits for detecting serum cytokines from breast cancer patients treated with immunotherapies. Furthermore, a small cytokine panel composed of CRP, PCT, and IL-6 is revealed to be significantly heterogeneous in each patient and highly dynamic for different treatment courses, showing promise as a panel of quantitative biomarker candidates for individual treatments. So, our work may provide a versatile diagnostic tool kit for the visual detection of clinical biomarkers with an adjustable broad detection range.

An electrochemical biosensor designed with entropy-driven autocatalytic DNA circuits for sensitive detection of ovarian cancer-derived exosomes.

Intelligent artificial DNA circuits have emerged as a promising approach for modulating signaling pathways and signal transduction through rational design, which may contribute to comprehensively realizing biomolecular sensing of organisms. In this work, we have fabricated an electrochemical biosensor for the sensitive and accurate detection of ovarian cancer-derived exosomes by constructing an entropy-driven autocatalytic DNA circuit (EADC). Specifically, the robust EADC is prepared by the self-assembly of well-designed DNA probes, and upon stimulation of the presence of ovarian cancer cells-derived exosomes, numerous inputs can be produced to feedback and accelerate the reaction. The catalytic abilities of the generated input sequences play a pivotal role in EADC and dramatically enhance the signal amplification capability. Through the combination of the autocatalytic circuit and circular cleavage reactions, significantly changed electrochemical signals can be recorded for sensitive analysis of the exosomes with a remarkably low detection limit of 30 particles/µL. Moreover, the proposed enzyme-free biosensor shows exceptional performance in distinguishing patient samples from healthy samples, which exhibits promising prospects for the clinical diagnosis of ovarian cancer.

An electrochemical biosensor to assay Trop-2 of breast cancer cells fabricated by methylene blue-assisted assembly of DNA nanoparticles.

Human trophoblast surface cell antigen 2 (Trop-2) on the tumor cell membrane can not only serve as the target for chemotherapy drugs, but also as a biomarker for typing and prognosis of breast cancer; however, assay of Trop-2 is seriously hampered due to the limitations of available tool. Herein, we have designed and fabricated an electrochemical biosensor for the assay of Trop-2 based on methylene blue (MB)-assisted assembly of DNA nanocomposite particles (DNPs). Specially, the recognition between Trop-2 and its aptamer may activate the primer exchange reaction (PER) on an electrode surface to produce long single-strand DNA (ssDNA) which can be self-assembled into DNPs by electrostatic interaction between negative charged DNA and positive charged and electro-active MB molecules which can also be used to give electrochemical signal. By using this electrochemical biosensor, ultrasensitive detection of tumor cells with high Trop-2 expressions can be conducted, with the limit of detection (LOD) of 1 cell/mL. Moreover, this biosensor can be further used for accurately profiling Trop-2 expression of tumor cells in mouse tissues, suggesting its great potential in the precise definition of breast cancer.

A peptides-based biosensor with target-triggered charge-switchable property for simple and sensitive detection of Granzyme B.

Granzyme B (GrB) is a serine protease released by natural killer cells and cytotoxic T lymphocytes during immune responses, which not only plays a role in tumor diagnosis but also provides valuable guidance during tumor treatment. In this work, we have designed a charge-switching peptide to fabricate an electrochemical biosensor for quantitative analysis of GrB. Specifically, the designed zwitterionic peptide is in an electrically neutral state before activation, and a door lock structure (proline) is constructed by utilizing the selectivity of carboxypeptidase A (CPA) to the carboxy-terminus of the peptide chain. The door lock is opened when the target is present, allowing CPA to hydrolyze the peptide. At this time, the peptide will convert from neutral to positive, triggering the assembly of a positively charged peptide layer on the electrode surface, resulting in a signal change. Studies have shown that the biosensor has good analytical performance, with a detection range of 0.01 pM-8 pM and a detection limit as low as 3.5 fM. Moreover, the developed biosensor has been effectively applied to the analysis of clinical samples, demonstrating its ability to monitor tumor progression and treatment with clinical applications.

An electrochemical biosensor to identify the phenotype of aggressive breast cancer cells.

Identifying the phenotype of aggressive breast cancer (BC) cells is vital for the effectiveness of surgical intervention and standard-of-care therapy. HER-2 is overexpressed in aggressive BC and MMP-2 is a crucial indicator of invasiveness and metastasis of BC, so we have proposed an electrochemical biosensor in this work to identify the phenotype of aggressive BC cells via detection of HER-2 together with MMP-2 by designing a dual-trapping peptide and a metal organic framework (MOF)-based probe. Specifically, the designed peptide contains both a HER-2 recognition sequence and MMP-2-specific substrate, while the MOF-based probe (AuNPs@HRP@ZIF-8), prepared by loading horseradish peroxidase (HRP) and gold nanoparticles (AuNPs) on ZIF-8, can also combine with the peptide. Consequently, sensitive and specific detection of both HER-2 and MMP-2 can be achieved in the wide range from 50 fg mL-1 to 50 ng mL-1 and 10 fg mL-1 to 10 ng mL-1, respectively, and the biosensor can distinguish HER-2+ BC cells and evaluate the invasion capability, which might be extended to provide a method for the accurate identification of tumor features in BC subtypes.

Application of functional peptides in the electrochemical and optical biosensing of cancer biomarkers.

Early screening and diagnosis are the most effective ways to prevent the occurrence and progression of cancers, thus many biosensing strategies have been developed to achieve economic, rapid, and effective detection of various cancer biomarkers. Recently, functional peptides have been gaining increasing attention in cancer-related biosensing due to their advantageous features of a simple structure, ease of synthesis and modification, high stability, and good biorecognition, self-assembly and antifouling capabilities. Functional peptides can not only act as recognition ligands or enzyme substrates for the selective identification of different cancer biomarkers but also function as interfacial materials or self-assembly units to improve the biosensing performances. In this review, we summarize the recent advances in functional peptide-based biosensing of cancer biomarkers according to the used techniques and the roles of peptides. Particular attention is focused on the use of electrochemical and optical techniques, both of which are the most commonly used techniques in the field of biosensing. The challenges and promising prospects of functional peptide-based biosensors in clinical diagnosis are also discussed.

An electrochemical biosensor for SARS-CoV-2 detection via its papain-like cysteine protease and the protease inhibitor screening.

The persistent coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is still infecting hundreds of thousands of people every day. Enriching the kits for SARS-CoV-2 detection and developing the drugs for patient treatments are still urgently needed for combating the spreading virus, especially after the emergence of various mutants. Herein, an electrochemical biosensor has been fabricated in this work for the detection of SARS-CoV-2 via its papain-like cysteine protease (PLpro) and the screening of protease inhibitor against SARS-CoV-2 by using our designed chimeric peptide-DNA (pDNA) nanoprobes. Utilizing this biosensor, the sensitive and specific detection of SARS-CoV-2 PLpro can be conducted in complex real environments including blood and saliva. Five positive and five negative patient throat swab samples have also been tested to verify the practical application capability of the biosensor. Moreover, we have obtained a detection limit of 27.18 fM and a linear detection range from 1 pg mL-1 to 10 µg mL-1 (I = 1.63 + 4.44 lgC). Meanwhile, rapid inhibitor screening against SARS-CoV-2 PLpro can be also obtained. Therefore, this electrochemical biosensor has the great potential for COVID-19 combating and drug development.

Engineering m6A demethylation-activated DNAzyme for visually and sensitively sensing fat mass and obesity-associated protein.

Fat mass and obesity-associated protein (FTO) regulating the N6-methyladenine (m6A, the most pervasive epigenetic modification) levels within the nucleus has been identified as a potential biomarker for cancer diagnosis and prognosis. However, current methods for FTO detection are complicated or/and not sensitive enough for practical application. Herein, we propose a colorimetric biosensor for detecting FTO based on a delicate design of m6A demethylation-activated DNAzyme. Specifically, an m6A-blocked DNAzyme is constructed as a switch of the biosensor that can be turned on by target FTO. The decreased thermal stability resulting from substrate cleavage leads to a DNAzyme recycling to produce multiple primers. Then the rolling circle amplification (RCA) reactions can be initiated to generate G-quadruplex-DNAzymes catalyzing 2,2-azino-bis-(3-ethylben-zthiazoline-6-sulfonic acid (ABTS) oxidation which can be readily observed by the naked eye. Quantitative detection can also be achieved with a limit of detection (LOD) down to 69.9 fM, exhibiting higher sensitivity than previous reports. Therefore, this biosensor opens a simple and sensitive way to achieve visual assay of FTO via triple signal amplification. In addition, our biosensor has been successfully applied to FTO detection in clinical samples, which shows great potential in clinical molecular diagnostics.

Functionalization of Covalent Organic Frameworks with Peptides by Polymer-Assisted Surface Modification and the Application for Protein Detection.

Although covalent organic frameworks (COFs) have received extensive attention for biomedical research due to their unique properties, their application is still hindered by the challenges of incorporating COFs with functional biomolecules. Since peptides have shown advantages in biomedical applications, herein, we propose the functionalization of COFs with peptides by a polymer-assisted surface modification strategy. Furthermore, a method based on the peptide-functionalized COFs for protein detection has also been developed to demonstrate their application potential. With the help of the polymers, peptides and horseradish peroxidase are attached onto COFs with a high surface density, and the developed method has achieved simple and sensitive detection of the secreted protein acidic and rich in cysteine. We speculate that the facile method proposed in this work to prepare peptide-functionalized COFs can not only benefit protein detection but also promote more biomedical applications of COFs.

Fabrication of a Polyvalent Aptamer Network on an Electrode Surface for Capture and Analysis of Circulating Tumor Cells.

Capture and analysis of circulating tumor cells (CTCs) from complex matrixes is pivotal for the prediction of cancer metastasis and personalized treatment of cancer. Herein, we propose a strategy for CTC capture by design and fabrication of a polyvalent aptamer network on an electrode surface, which can be further used for the sensitive analysis of CTCs. In our design, the polyvalent aptamer network, which is constructed via a rolling circle amplification reaction, can significantly enhance the cell-binding abilities. Meanwhile, tetrahedral DNA structures previously assembled on the electrode surface will promote the spatial orientation and reduce the steric hindrance effect of the cell capture, thus improving the cell capture efficiency. Importantly, a detectable electrochemical signal can be obtained without additional signal probes by means of target-induced allostery of the DNA hairpin structures. Further studies reveal that the electrochemical response is proportional to the logarithm of the CTC abundance ranging from 102 to 5 × 104 cell mL-1 with a low limit of detection of 23 cell mL-1. Moreover, the proposed capture strategy exhibits excellent stability and anti-interference in human whole blood, indicating its promising potential in clinical diagnosis.

Covalent organic frameworks (COFs)-based biosensors for the assay of disease biomarkers with clinical applications.

Covalent organic frameworks (COFs) are an emerging type of porous crystalline polymers that are built by light elements (typically H, B, C, N, O and Si) via organic covalent bonds. Currently, COFs have been exploited for biomedical application due to their unique properties, such as structural diversity, intrinsic stability, ordered porosity, tailor-made functions, and excellent adsorption features. In particular, COFs are increasingly popular in the construction of biosensors for the detection of various disease biomarkers, and have been extended to the clinical applicability for early diagnostics, medication instruction and prognostic monitoring of diseases. In this review, we mainly summarize the recent advances on COFs-based biosensors for the assay of disease biomarkers with clinical applications. According to the features of molecular structure, disease biomarkers are classified into four categories, including small biological ions/molecules, proteins, nucleic acids, and cancer cells/exosomes. Impressively, COFs-based biosensors present a bright prospect in clinical diagnosis of diseases in both hospital-end and household-end utilization.

An electrochemical biosensor based on electroactive peptide nanoprobes for the sensitive analysis of tumor cells.

Peptides possess many appealing and desirable features, which have attracted increasing attention in the field of electrochemical biosensing. However, peptides hardly produce noticeable electronic signals in response to target binding events. In this work, amphipathic peptides FFFGGGGRGDS with both target recognition and self-assembly capabilities are designed to be co-assembled with the electroactive species ferrocenecarboxylic acid (FcCOOH). Furthermore, the resultant electroactive peptide nanoprobes (ePNPs) are applied for sensitive electrochemical analysis of tumor cells. Specifically, tumor cells are captured by the electrode modified with the corresponding DNA aptamers, and ePNPs can then selectively bind to integrin proteins on the cell surface, thereby accompanied by a remarkable increase of electrochemical signal. Taking the assay of MDA-MB-231 cells, the fabricated biosensor can detect cancer cells with a detection limit of 7 cells mL-1. Moreover, the ePNPs can act as a universal probe for the detection of different cell lines. Given the merits of easy synthesis, convenient operation, and favorable analytical performance, the proposed biosensor exhibits great potential in developing peptide-based electrochemical biosensing for clinical applications.

Molecular Characterization of Exosomes for Subtype-Based Diagnosis of Breast Cancer.

Breast cancer is very heterogeneous and the most frequently diagnosed cancer worldwide, and precise therapy targeting specific subtypes may improve the survival rates of breast cancer patients. In this study, we designed a biomimetic vesicle by camouflaging catalytic DNA machinery with a breast cancer cell membrane, which enabled the molecular classification of circulating exosomes for subtype-based diagnosis through homotypic recognition. In addition, the vesicles specifically targeted and fused with breast cancer exosomes with phenotypic homology and manipulated the DNA machinery to amplify electrochemical signaling using exosomal RNA as an endogenous trigger. The biomimetic vesicles prepared with MCF-7 cancer cell-derived membranes were shown to recognize estrogen receptor-positive breast cancer exosomes and exhibited a low detection limit of 557 particles mL-1 with microRNA-375 used as the endogenous biomarker. Furthermore, the biomimetic vesicles prepared with MDA-MB-231 cancer cell-derived membranes displayed satisfactory performance in a homotypic analysis of triple-negative breast cancer exosomes with a potential therapeutic target, PD-L1 mRNA, used as the endogenous biomarker. Most importantly, cross-validation experiments confirmed the high accuracy and selectivity of this homotypic recognition-driven analysis for molecular subtyping of breast cancer. When applied to clinical samples of breast cancer patients, the vesicles demonstrated feasibility and reliability for evaluating the molecular features of cancer cell-derived exosomes and enabled stage-specific monitoring of breast cancer patients because the electrochemical signals showed a positive correlation with disease progression. Therefore, this work may provide new ideas for the precise diagnosis and personalized treatment of breast cancer patients throughout the whole disease process.

Electrochemical Evaluation of Tumor Development via Cellular Interface Supported CRISPR/Cas Trans-Cleavage.

Evaluating tumor development is of great importance for clinic treatment and therapy. It has been known that the amounts of sialic acids on tumor cell membrane surface are closely associated with the degree of cancerization of the cell. So, in this work, cellular interface supported CRISPR/Cas trans-cleavage has been explored for electrochemical simultaneous detection of two types of sialic acids, i.e., N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac). Specifically, PbS quantum dot-labeled DNA modified by Neu5Gc antibody is prepared to specifically recognize Neu5Gc on the cell surface, followed by the binding of Neu5Ac through our fabricated CdS quantum dot-labeled DNA modified by Sambucus nigra agglutinin. Subsequently, the activated Cas12a indiscriminately cleaves DNA, resulting in the release of PbS and CdS quantum dots, both of which can be simultaneously detected by anodic stripping voltammetry. Consequently, Neu5Gc and Neu5Ac on cell surface can be quantitatively analyzed with the lowest detection limits of 1.12 cells/mL and 1.25 cells/mL, respectively. Therefore, a ratiometric electrochemical method can be constructed for kinetic study of the expression and hydrolysis of Neu5Gc and Neu5Ac on cell surface, which can be further used as a tool to identify bladder cancer cells at different development stages. Our method to evaluate tumor development is simple and easy to be operated, so it can be potentially applied for the detection of tumor occurrence and development in the future.