Magnetic Nanoagent Coated with an Activated Macrophage Membrane for Colorimetric Detection of Bacteria.

The construction of cell mimics replicating the surface landscape and biological functions of the cell membrane offers promising prospects for biomedical research and applications. Inspired by the inherent recognition capability of immune cells toward pathogens, we have fabricated activated macrophage membrane-coated magnetic silicon nanoparticles (aM-MSNPs) in this work as an isolation and recognition tool for enhanced bacterial analysis. Specifically, the natural protein receptors on the activated macrophage membrane endow the MSNPs with a broad-spectrum binding capacity to different pathogen species. By further incorporation of a tyramide amplification strategy, direct naked-eye analysis of specific bacteria with a detection limit of 10 CFU/mL can be achieved. Moreover, application to the diagnosis of urinary tract infections has also been validated, and positive samples spiked with bacteria can be clearly distinguished with an accuracy of 100%. This work may enrich cell membrane-based architectures and provide an experimental paradigm for point-of-care testing (POCT) detection of bacteria.

Synthesis of a COF-on-MOF hybrid nanomaterial for enhanced colorimetric biosensing.

The construction of hybrid materials is significant for the exploration of functionalities in colorimetric biosensing due to its structural designability and synergy effects. In this work, a COF-on-MOF hybrid nanomaterial has been newly synthesized for colorimetric biosensing. Experimental results reveal that on-surface synthesis of COF on MOF brings nanoscale proximity between COF and MOF, which exhibits more than two folds of peroxidase-like activity as compared to single Fe-MOF. Therefore, by using the MCA@Fe-MOF nanomaterial with the assist of a specific acetyl-peptide, MCA@Fe-MOF can serve as an efficient signal reporter for colorimetric assay of histone deacetylase (HDAC), and the limit of detection (LOD) can be as low as 0.261 nM. Looking forward, the demand for diverse and promising COF-on-MOF nanomaterials with varied functionalities is anticipated, propelling further exploration of their role in colorimetric biosensing.

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.

Enhanced performance of enzymes confined in biocatalytic hydrogen-bonded organic frameworks for sensing of glutamate in the central nervous system.

Glutamate (Glu) is a key excitatory neurotransmitter associated with various neurological disorders in the central nervous system, so its measurement is vital to both basic research and biomedical application. In this work, we propose the first example of using biocatalytic hydrogen-bonded organic frameworks (HOFs) as the hosting matrix to encapsulate glutamate oxidase (GLOD) via a de novo approach, fabricating a cascaded-enzyme nanoreactor for Glu biosensing. In this design, the ferriporphyrin ligands can assemble to form Fe-HOFs with high catalase-like activity, while offering a scaffold for the in-situ immobilization of GLOD. Moreover, the formed GLOD@Fe-HOFs are favorable for the efficient diffusion of Glu into the active sites of GLOD via the porous channels, accelerating the cascade reaction with neighboring Fe-HOFs. Consequently, the constructed nanoreactor can offer superior activity and operational stability in the catalytic cascade for Glu biosensing. More importantly, rapid and selective detection can be achieved in the cerebrospinal fluid (CSF) collected from mice in a low sample consumption. Therefore, the successful fabrication of enzyme@HOFs may offer promise to develop high-performance biosensor for further biomedical applications.

Preparation of Well-Constructed and Metal-Modified Covalent Organic Framework Nanoparticles for Biosensing Design with Cascade Catalytic Capability.

Uniform covalent organic framework nanoparticles (COF NPs) with a well-defined pore structure may provide a robust platform for scaffolding enzymes. Herein, bipyridine-based spherical COF NPs have been successfully prepared in this work through the Schiff base condensation reaction. Moreover, they are functionalized by metal modification and are further used for biosensor fabrication. Experimental results reveal that the metal-modified COF NPs also display impressive peroxidase-like catalytic activities, while they can load enzymes, such as glucose oxidase (GOx) and sarcosine oxidase (SOx), to develop a cascade catalysis system for design of various kinds of biosensors with very well performance. For example, the optimized GOx@Fe-COFs can achieve a sensitive detection of glucose with a low limit of detection (LOD) of 12.8 µM. Meanwhile, the enzymes also exhibit a commendable preservation of 80% enzymatic activity over a span of 14 days under ambient conditions. This work may pave the way for advancing cascade catalysis and the analysis of different kinds of biological molecules based on COF NPs.

Graphene Oxide-Mediated Regulation of Volume Exclusion and Wettability in Biomimetic Phosphorylation-Responsive Ionic Gates.

Replicating phosphorylation-responsive ionic gates via artificial fluidic systems is essential for biomolecular detection and cellular communication research. However, current approaches to governing the gates primarily rely on volume exclusion or surface charge modulation. To overcome this limitation and enhance ion transport controllability, we introduce graphene oxide (GO) into nanochannel systems, simultaneously regulating the volume exclusion and wettability. Moreover, inspired by (cAMP)-dependent protein kinase A (PKA)-regulated L-type Ca2+ channels, we employ peptides for phosphorylation which preserves them as nanoadhesives to coat nanochannels with GO. The coating boosts steric hindrance and diminishes wettability, creating a substantial ion conduction barrier, which represents a significant advancement in achieving precise ion transport regulation in abiotic nanochannels. Leveraging the mechanism, we also fabricated a sensitive biosensor for PKA activity detection and inhibition exploration. The combined regulation of volume exclusion and wettability offers an appealing strategy for controlled nanofluidic manipulation with promising biomedical applications in diagnosis and drug discovery.

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.

Engineered Escherichia coli as a Controlled-Release Biocarrier for Electrochemical Immunoassay.

Micro/nanocarriers hold great potential in bioanalysis for molecular recognition and signal amplification but are frequently hampered by harsh synthesis conditions and time-consuming labeling processes. Herein, we demonstrate that Escherichia coli (Ec) can be engineered as an efficient biocarrier for electrochemical immunoassay, which can load ultrahigh amounts of redox indicators and simultaneously be decorated with detection antibodies via a facile polydopamine (PDA)-mediated coating approach. Compared with conventional carrier materials, the entire preparation of the Ec biocarrier is simple, highly sustainable, and reproducible. Moreover, immune recognition and electrochemical transduction are performed independently, which eliminates the accumulation of biological interference on the electrode and simplifies electrode fabrication. Using human epidermal growth factor receptor 2 (HER2) as the model target, the proposed immunosensor exhibits excellent analytical performance with a low detection limit of 35 pg/mL. The successful design and deployment of Ec biocarrier may provide new guidance for developing biohybrids in biosensing 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.

Dual-Gene-Controlled Rolling Circle Amplification Strategy for SARS-CoV-2 Analysis.

The development of sensitive, accurate, and conveniently operated methods for the simultaneous assay of two nucleic acids is promising while still challenging. In this work, by using two genes (the N gene and RdRp gene) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as examples, we have designed an ingenious dual-gene-controlled rolling circle amplification (RCA) strategy to propose an accurate and sensitive electrochemical method. Specifically, the coexistence of the two target genes can trigger the RCA reaction to generate a number of repeated G-quadruplex (G4)-forming sequences. These sequences then switch into G4/hemin complexes with redox activity after the incubation of hemin, which can catalyze the TMB/H2O2 substrates to produce significantly enhanced current responses. Experimental results reveal that the proposed method exhibits satisfying feasibility and analytical performance, enabling the sensitive detection of SARS-CoV-2 in the range of 0.1-5000 pM, with the detection limit of 57 fM. Meanwhile, because only the simultaneous existence of the two target genes can effectively trigger the downstream amplification reaction, this method can effectively avoid false-positives and ensure specificity as well as accuracy. Furthermore, our method can distinguish the COVID-19 samples from healthy people, and the outcomes show a satisfying agreement with the results of RT-PCR, manifesting that our label-free dual-gene-controlled RCA strategy exhibits great possibility in clinical application.

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.

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.

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.

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.

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.

Hydrazone chemistry-mediated CRISPR/Cas12a system for bacterial analysis.

In this study, a hydrazone chemistry-mediated clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system has been proposed for the fist time and constructed. In our system, hydrazone chemistry is designed and employed to accelerate the formation of a whole activation strand by taking advantage of the proximity effect induced by complementary base pairing, thus activating the CRISPR/Cas12a system quickly and efficiently. Moreover, the introduction of hydrazone chemistry can improve the specificity of the CRISPR/Cas12a system, allowing it to effectively distinguish single-base mismatches. The established system has been further applied to analyze Pseudomonas aeruginosa by specific recognition of the probe strand with a characteristic fragment in 16S rDNA to release the hydrazine group-modified activation strand. The method shows a wide linear range from 3.8 × 102 colony-forming units (CFU)/ml to 3.8 × 106 CFU/ml, with the lowest detection limit of 24 CFU/ml. Therefore, the introduction of hydrazone chemistry may also broaden the application of the CRISPR/Cas12a system.