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Minimal residual disease (MRD) provides an independent prognostic factor for multiple myeloma (MM) patients. However, clinical MRD assays suffer from highly invasive sampling, insufficient detection sensitivity, and high cost. Herein, a stiMulus-Responsive ligand-Decorated microfluidic chip (MRD-Chip) was developed for efficient capture and controlled release of circulating myeloma cells (CMCs) in the peripheral blood for noninvasive myeloma evaluation. The CD138 antibody-decorated herringbone chip with a disulfide linker was designed to enhance the collision probability between blood cells and capture antibodies, leading to high capture efficiency of CMCs. More importantly, the captured CMCs can be nondestructively released via a thiol-exchange reaction, allowing them to be used for subsequent cellular and molecular analysis. By fluorescence in situ hybridization assay, we successfully identified the cytogenetic abnormalities (chromosome 1q21 amplification and p53 deletion) of CMCs in clinical samples. Overall, with the merits of noninvasive sampling, high capture efficiency (70.93%), high throughput (1.5 mL/h), and nondestructive release of target cells (over 90% viability) for downstream analysis, our strategy provides new opportunities for myeloma evaluation, such as prognosis assessment, efficacy monitoring, and mechanism research of disease relapse and drug resistance.

The past decade has witnessed ongoing progress in precision medicine to improve human health. As an emerging diagnostic technique, liquid biopsy can provide real-time, comprehensive, dynamic physiological and pathological information in a noninvasive manner, opening a new window for precision medicine. Liquid biopsy depends on the sensitive and reliable detection of circulating targets (e.g., cells, extracellular vesicles, proteins, microRNAs) from body fluids, the performance of which is largely governed by recognition ligands. Aptamers are single-stranded functional oligonucleotides, capable of folding into unique tertiary structures to bind to their targets with superior specificity and affinity. Their mature evolution procedure, facile modification, and affinity regulation, as well as versatile structural design and engineering, make aptamers ideal recognition ligands for liquid biopsy. In this review, we present a broad overview of aptamer-based liquid biopsy techniques for precision medicine. We begin with recent advances in aptamer selection, followed by a summary of state-of-the-art strategies for multivalent aptamer assembly and aptamer interface modification. We will further describe aptamer-based micro-/nanoisolation platforms, aptamer-enabled release methods, and aptamer-assisted signal amplification and detection strategies. Finally, we present our perspectives regarding the opportunities and challenges of aptamer-based liquid biopsy for precision medicine.

New neutralizing agents against SARS-CoV-2 and the associated mutant strains are urgently needed for the treatment and prophylaxis of COVID-19. Herein, we develop a spherical cocktail neutralizing aptamer-gold nanoparticle (SNAP) to synergistically block the interaction of SARS-CoV-2 receptor-binding domain (RBD) and angiotensin-converting enzyme-2 (ACE2). Taking advantage of the simultaneous recognition of multi-homologous and multi-heterogenous neutralizing aptamers and dimensionally matched nano-scaffolds, the SNAP exhibits increased affinity to the RBD with a dissociation constant value of 5.46 pM and potent neutralization against authentic SARS-CoV-2 with a half-maximal inhibitory concentration of 142.80 aM. Additional benefits include the multi-epitope blocking capability of the aptamer cocktail and the steric hindrance of the nano-scaffold, which further covers the ACE2 binding interfaces and affects the conformational transition of the spike protein. As a result, the SNAP strategy exhibits broad neutralizing activity, almost completely blocking the infection of N501Y and D614G mutant strains. Overall, the SNAP strategy provides a new direction for development of anti-virus infection mechanisms, both to fight the COVID-19 pandemic and serve as a powerful technical reserve for future unknown pandemics.

Based on the linkage of genotype and phenotype, display technology has been widely used to generate specific ligands for profiling, imaging, diagnosis and therapy applications. However, due to the lack of effective monoclonal manipulation and affinity evaluation methods, traditional display technology has to undergo tedious steps of selection, clone isolation, amplification, sequencing, synthesis and characterization to obtain the binding sequences. To directly acquire high-affinity clones, we propose a double monoclonal display approach (dm-Display) for peptide screening based on highly paralleled monoclonal manipulation in emulsion droplets. dm-Display can monoclonally link the genotype, phenotype and affinity to realize integrated monoclonal separation, amplification, recognition and staining in one droplet so that discrete high-affinity clones can be quickly extracted. Monoclonal manipulations highly-parallelly occur in millions of droplets so that molecular screening of a highly diverse phage library is achieved. We have screened specific peptide ligands against CD71 and GPC1, proving the feasibility and generality of dm-Display. As a highly efficient ligand screening platform, dm-Display will promote the further development of molecular screening.

In comparison to mouse, the developmental process of human islets has not been properly elucidated. The advancement of single cell RNA-seq technology enables us to study the properties of alpha and beta cells at single cell resolution. By using mitochondrial genome variants as endogenous lineage-tracing markers, we found that human alpha and beta cells have different lineage features. This finding suggests specific endocrine progenitors for alpha and beta cells, which is different from mouse islet cells. This strategy was also applied to a study of chemically-induced islet cell reprogramming and was used to help identify artemether-induced alpha-to-beta trans-differentiation in human islets. The computational results of this study will inspire future studies to establish, maintain, and expand beta cell-specific progenitors in vitro and in vivo.

An aqueous two-phase system (ATPS) is a water-in-water biphasic system, which is generally formed by two incompatible polymers. Recently, considerable effort has been dedicated to search for new ATPS polymer pairs to further expand ATPS's applications. In this paper, a new ATPS system based on silk fibroin (SF) and alginate is introduced. A phase diagram was established to show the critical concentrations for the formation of an SF/alginate ATPS. The present system is sensitive to pH stimulus and transformed from an ATPS into a single-phasic system as pH increases above ∼9.5. Circular dichroism, fluorescence emission spectra, hydrodynamic diameter, and ζ-potential data together indicate that the SF chains undergo a dramatic extension as pH is increased, which is the reason underlying the pH-triggered phase transition. As feasible applications of this biphasic system, compartmentalized multiplex immunoassay, controlled encapsulation and release, and hierarchical fiber fabrication were demonstrated using the SF/alginate ATPS.

Liquid biopsy offers non-invasive and real-time molecular profiling of individual patients, and is thus considered a revolutionary technology in precision medicine. Exosomes have been acknowledged as significant biomarkers in liquid biopsy, as they play a central role in cell-cell communication and are closely related to the pathogenesis of most human malignancies. Nevertheless, in biofluids exosomes always co-exist with other particles, and the cargo components of exosomes are highly heterogeneous. Thus, the isolation and molecular characterization of exosomes are still technically challenging. Microfluidics technology effectively addresses this challenge by virtue of its inherent advantages, such as precise manipulation of fluids, low consumption of samples and reagents, and a high level of integration. Recent advances in microfluidics allow in situ exosome capture and molecular detection with unprecedented selectivity and sensitivity. In this review, the state-of-the-art developments in microfluidics-based exosome research, including exosome isolation approaches and molecular detection strategies, with highlights of the characterization of exosomal biomarkers in cancer liquid biopsy is summarized. The major challenges are also discussed and some perspectives for the future directions of exosome-based liquid biopsy in microfluidic systems are presented.

Tumor-derived exosomal proteins have emerged as promising biomarkers for cancer diagnosis, but the quantitation accuracy is hindered by large numbers of normal cell-derived exosomes. Herein, we developed a dual-target-specific aptamer recognition activated in situ connection system on exosome membrane combined with droplet digital PCR (ddPCR) (TRACER) for quantitation of tumor-derived exosomal PD-L1 (Exo-PD-L1 ). Leveraging the high binding affinity of aptamers, excellent selectivity of dual-aptamer recognition, and the high sensitivity of ddPCR, this method exhibits significant sensitivity and selectivity for tracing tumor-derived Exo-PD-L1 in a wash-free manner. Due to the excellent sensitivity, the level of tumor-derived Exo-PD-L1 detected by TRACER can distinguish cancer patients from healthy donors, and for the first time was identified as a more reliable tumor diagnostic marker than total Exo-PD-L1 . The TRACER strategy holds great potential for converting exosomes into reliable clinical indicators and exploring the biological functions of exosomes.

Digital-WGS enables high-performance single-cell sequencing with automatic processing based on digital microfluidics., Single-cell whole-genome sequencing (WGS) is critical for characterizing dynamic intercellular changes in DNA. Current sample preparation technologies for single-cell WGS are complex, expensive, and suffer from high amplification bias and errors. Here, we describe Digital-WGS, a sample preparation platform that streamlines high-performance single-cell WGS with automatic processing based on digital microfluidics. Using the method, we provide high single-cell capture efficiency for any amount and types of cells by a wetted hydrodynamic structure. The digital control of droplets in a closed hydrophobic interface enables the complete removal of exogenous DNA, sufficient cell lysis, and lossless amplicon recovery, achieving the low coefficient of variation and high coverage at multiple scales. The single-cell genomic variations profiling performs the excellent detection of copy number variants with the smallest bin of 150 kb and single-nucleotide variants with allele dropout rate of 5.2%, holding great promise for broader applications of single-cell genomics.

Magnetic resonance (MR) technology has been widely employed in scientific research, clinical diagnosis and geological survey. However, the fabrication of MR radio frequency probeheads still face difficulties in integration, customization and miniaturization. Here, we utilized 3D printing and liquid metal filling techniques to fabricate integrative radio frequency probeheads for MR experiments. The 3D-printed probehead with micrometer precision generally consists of liquid metal coils, customized sample chambers and radio frequency circuit interfaces. We screened different 3D printing materials and optimized the liquid metals by incorporating metal microparticles. The 3D-printed probeheads are capable of performing both routine and nonconventional MR experiments, including in situ electrochemical analysis, in situ reaction monitoring with continues-flow paramagnetic particles and ions separation, and small-sample MR imaging. Due to the flexibility and accuracy of 3D printing techniques, we can accurately obtain complicated coil geometries at the micrometer scale, shortening the fabrication timescale and extending the application scenarios., Here, the authors combine 3D printing and liquid metal filling techniques to fabricate customised probeheads for magnetic resonance experiments. They demonstrate in situ electrochemical nuclear magnetic resonance analysis, reaction monitoring with continues-flow separation and small-sample imaging.

As a malignant disease that seriously threatens human health, hepatocellular carcinoma (HCC) lacks effective early screening and prognostic assessment methods. Herein, we developed a method for efficient capture and multiphenotype analysis of circulating tumor cells (CTCs) of hepatocellular carcinoma. The anti-ASGPR antibody and the anti-EpCAM antibody were modified in parallel on a deterministic lateral displacement (DLD)-patterned microfluidic Synergetic-Chip to enhance capture efficiency by a complementary effect. CTCs were detected in 45 out of 45 (100%) HCC patients, with a sensitivity and specificity of 97.8 and 100%, respectively. Patients with more total CTCs and nonepithelial CTCs were in later stages of HCC and had more malignant progression. This strategy proposes a feasible approach for early diagnosis and prognosis of hepatocellular carcinoma.

Here, a fully integrated multicolor immunosensor was developed for sensitive and reliable semiquantitative analysis of HIV-1 p24, which integrates the multistep reactions of horseradish peroxidase (HRP)-linked immunoassay and gold nanorod (AuNR)-based multicolor assay into a single microfluidic chip. The HRP-linked immunoassay functions by moving magnetic beads bound to a capture antibody through different aqueous phases containing immunoassay reagents. HRP-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) is used to mediate AuNRs etching for producing various color changes. Multiple etching processes can be activated by simple mixing of the reagents from the reagent storage reservoir. The fully integrated strategy with sample-in answer-out capability is initiated by simple chip manipulation and finally concluded by converting recognition of antigen-antibody into a vivid color variation for direct visualization and semiquantitative analysis. By bare eye observation, our integrated multicolor immunosensor allows sensitive and reliable semiquantitative analysis of HIV-1 p24 within 1 h. The microfluidic chip device demonstrated here simplifies the operation significantly and thus allows broader application of a multicolor immunosensor for point of care (POC) testing in low-resource settings.

Nucleic acids are natural biopolymers of nucleotides that store, encode, transmit and express genetic information, which play central roles in diverse cellular events and diseases in living things. The analysis of nucleic acids and nucleic acids-based analysis have been widely applied in biological studies, clinical diagnosis, environmental analysis, food safety and forensic analysis. During the past decades, the field of nucleic acids analysis has been rapidly advancing with many technological breakthroughs. In this review, we focus on the methods developed for analyzing nucleic acids, nucleic acids-based analysis, device for nucleic acids analysis, and applications of nucleic acids analysis. The representative strategies for the development of new nucleic acids analysis in this field are summarized, and key advantages and possible limitations are discussed. Finally, a brief perspective on existing challenges and further research development is provided.

The World Health Organization has declared the outbreak of a novel coronavirus (SARS-CoV-2 or 2019-nCoV) as a global pandemic. However, the mechanisms behind the coronavirus infection are not yet fully understood, nor are there any targeted treatments or vaccines. In this study, we identified high-binding-affinity aptamers targeting SARS-CoV-2 RBD, using an ACE2 competition-based aptamer selection strategy and a machine learning screening algorithm. The Kd values of the optimized CoV2-RBD-1C and CoV2-RBD-4C aptamers against RBD were 5.8 nM and 19.9 nM, respectively. Simulated interaction modeling, along with competitive experiments, suggests that two aptamers may have partially identical binding sites at ACE2 on SARS-CoV-2 RBD. These aptamers present an opportunity for generating new probes for recognition of SARS-CoV-2 and could provide assistance in the diagnosis and treatment of SARS-CoV-2 while providing a new tool for in-depth study of the mechanisms behind the coronavirus infection.

Single-cell RNA sequencing (scRNA-seq) is a powerful method in investigating single-cell heterogeneity to reveal rare cells, identify cell subpopulations, and construct a cell atlas. Conventional benchtop methods for scRNA-seq, including multistep operations, are labor intensive, reaction inefficient, contamination prone, and reagent consuming. Here we report a digital microfluidics-based single-cell RNA sequencing (digital-RNA-seq) for simple, efficient, and low-cost single-cell mRNA measurements. Digital-RNA-seq automates fluid handling as discrete droplets to sequentially perform protocols of scRNA-seq. To overcome the current problems of single-cell isolation in efficiency, integrity, selectivity, and flexibility, we propose a new strategy, passive dispensing method, relying on well-designed hydrophilic-hydrophobic microfeatures to rapidly generate single-cell subdroplets when a droplet of cell suspension is encountered. For sufficient cDNA generation and amplification, digital-RNA-seq uses nanoliter reaction volumes and hydrophobic reaction interfaces, achieving high sensitivity in gene detection. Additionally, the stable droplet handling and oil-closed reaction space featured in digital-RNA-seq ensure highly accurate measurement. We demonstrate the functionality of digital-RNA-seq by quantifying heterogeneity among single cells, where digital-RNA-seq shows excellent performance in rare transcript detection, cell type differentiation, and essential gene identification. With the advantages of automation, sensitivity, and accuracy, digital-RNA-seq represents a promising scRNA-seq platform for a wide variety of biological applications.

The World Health Organization has declared the outbreak of a novel coronavirus (SARS-CoV-2 or 2019-nCoV) as a global pandemic. However, the mechanisms behind the coronavirus infection are not yet fully understood, nor are there any targeted treatments or vaccines. In this study, we identified high-binding-affinity aptamers targeting SARS-CoV-2 RBD, using an ACE2 competition-based aptamer selection strategy and a machine learning screening algorithm. The Kd values of the optimized CoV2-RBD-1C and CoV2-RBD-4C aptamers against RBD were 5.8 nM and 19.9 nM, respectively. Simulated interaction modeling, along with competitive with experiments, suggests that two aptamers may have partially identical binding sites at ACE2 on SARS-CoV-2 RBD. These aptamers present an opportunity for generating new probes for recognition of SARS-CoV-2, and could provide assistance in the diagnosis and treatment of SARS-CoV-2 while providing a new tool for in-depth study of the mechanisms behind the coronavirus infection.

ScRNA-seq has the ability to reveal accurate and precise cell types and states. Existing scRNA-seq platforms utilize bead-based technologies uniquely barcoding individual cells, facing practical challenges for precious samples with limited cell number. Here, we present a scRNA-seq platform, named Paired-seq, with high cells/beads utilization efficiency, cell-free RNAs removal capability, high gene detection ability and low cost. We utilize the differential flow resistance principle to achieve single cell/barcoded bead pairing with high cell utilization efficiency (95%). The integration of valves and pumps enables the complete removal of cell-free RNAs, efficient cell lysis and mRNA capture, achieving highest mRNA detection accuracy (R = 0.955) and comparable sensitivity. Lower reaction volume and higher mRNA capture and barcoding efficiency significantly reduce the cost of reagents and sequencing. The single-cell expression profile of mES and drug treated cells reveal cell heterogeneity, demonstrating the enormous potential of Paired-seq for cell biology, developmental biology and precision medicine., Single-cell RNA-seq can reveal accurate and precise cell types and states. Here the authors present an scRNA-seq platform, Paired-seq, which uses differential flow resistance to achieve 95% cell utilisation efficiency for improved cell-free RNA removal and gene detection.

The dissociation constant (Kd) is a crucial parameter for characterizing binding affinity in molecular recognition, including antigen-antibody, DNA-protein, and receptor-ligand interactions. However, conventional methods for Kd characterization usually involve a multi-step process and time-consuming operations for incubation, washing, and detection, thus causing problems, such as time delays, microbead loss, degradation of sensitive molecules, and personal errors. Here we demonstrate an automated ligand binding affinity evaluation platform (Auto-affitech) using digital microfluidics (DMF), with individual droplets at the microliter level, programmed to rapidly perform the incubation and separation of target-beads and binding ligands. Because the loss of the beads influences the detection results, we propose a new strategy for magnetic bead separation using DMF, termed the bidirectional separation method. By splitting one droplet into two asymmetric droplets, high bead retention efficiency (89.57% ± 0.05%) and high washing efficiency (99.59% ± 0.17%, with four washings) were obtained. We demonstrate the determination of Kd of an aptamer-protein system (EpCAM and its corresponding aptamer SYL3C) and an antigen-antibody system (H5N1 antigen and antibody), proving the capability and universality of Auto-affitech in various receptor-ligand systems. Integrating all the sample processing procedures, the Auto-affitech not only saves manual labor and minimizes personal errors, but also conserves samples and shortens analysis time. Overall, this platform successfully demonstrates to be an automated approach for dissociation constant evaluation and exhibits great potential for highly efficient screening of ligands.

Circulating tumor cells (CTCs) undergoing epithelial mesenchymal transition (EMT) play an essential role in metastasis and have a better correlation with poor disease outcomes, but the most current affinity-based enrichment methods rely on targeting epithelial markers, which are less effective in capturing CTCs undergoing EMT. Herein, we identified and optimized an aptamer (ZY5C) sequence with high binding affinity and specificity against cell surface vimentin (CSV), which is overexpressed on the post-EMT CTCs. Not only can the hairpin-structured ZY5C aptamer specifically recognize a number of cancer cells with native CSV expression, but it can also bind to CSV expressed on EMT-cells. The Kd value of the ZY5C aptamer against CSV-positive T24 cells was found to be 38 ± 4 nM. Using the evolved ZY5C aptamer and multivalent ZY5C aptamer-functionalized chip, we were able to isolate CTCs from the blood of adenocarcinoma, sarcoma, and carcinosarcoma patients. Overall, this ZY5C aptamer and isolation method bring a fresh approach to CTCs analysis, which not only detects CTCs from nonepithelial origin, but also provides an efficient way to in-depth study the role of post-EMT CTCs in clinical application and metastasis mechanisms.

Minimal residual disease (MRD) offers a highly independent prognostic factor for leukemia patients. However, challenges confronting conventional MRD assays are high invasiveness, as well as limited detection sensitivity and clinical applicability. Inspired by the self-adaptive skeleton and multiple suckers or tendrils of climbing plants, a biomimetic Multivalent Aptamer Nanoclimber (MANC)-functionalized microfluidic chip (MANC-Chip) is reported for minimally invasive, highly sensitive and clinically applicable MRD detection in the peripheral blood of T-cell acute lymphoblastic leukemia patients. The MANCs are synthesized by a simple co-polymerization reaction. Due to their flexible structure and cooperative multivalent effect, MANCs dramatically enhance the binding affinity of aptamers targeting leukemia cells. A deterministic lateral displacement-patterned microfluidic chip is designed to further increase the collision probability between MANCs and leukemia cells. Benefiting from the synergistic effect of multivalent binding and enhanced collision, a high capture efficiency of 92.2% for leukemia cells is achieved. Moreover, the captured leukemia cells can be released with high efficiency of 88.9% and high viability of 93.8% via nuclease treatment prior to downstream analysis. Overall, the excellent features of MANC-Chip make it very useful for precise detection of MRD and better understanding of leukemia.

The ubiquitous biomembrane interface, with its dynamic lateral fluidity, allows membrane-bound components to rearrange and localize for high-affinity multivalent ligand-receptor interactions in diverse life activities. Inspired by this, we herein engineered a fluidic multivalent nanointerface by decorating a microfluidic chip with aptamer-functionalized leukocyte membrane nanovesicles for high-performance isolation of circulating tumor cells (CTCs). This fluidic biomimetic nanointerface with active recruitment-binding afforded significant affinity enhancement by 4 orders of magnitude, exhibiting 7-fold higher capture efficiency compared to a monovalent aptamer functionalized-chip in blood. Meanwhile, this soft nanointerface inherited the biological benefits of a natural biomembrane, minimizing background blood cell adsorption and maintaining excellent CTC viability (97.6%). Using the chip, CTCs were successfully detected in all cancer patient samples tested (17/17), suggesting the high potential of this fluidity-enhanced multivalent binding strategy in clinical applications. We expect this bioengineered interface strategy will lead to the design of innovative biomimetic platforms in the biomedical field by leveraging natural cell-cell interaction with a natural biomaterial.

Objective To ascertain whether en bloc resection could reduce the risk of seeding cancer cells into the circulation during the resection of non-muscle invasive bladder cancer (NMIBC). Methods Patients with primary NMIBC were enrolled in this prospective study from October 2017 to May 2018. Patients were allocated to receive conventional transurethral resection of the bladder (TURB) or retrograde en bloc resection technique of the bladder tumor (RERBT). Blood samples (1 ml) for circulating tumor cell (CTC) enumeration were drawn from the peripheral vein prior to resection (PV1), immediately after resection of the tumor base (PV2), and at 12 h after resection (PV3). Intra-group comparisons of the changes in the number of CTCs identified among the PV1, PV2, and PV3 blood samples were performed in each group. Results A total of 21 patients (12 in the RERBT group and 9 in the TURB group) were recruited. For patients receiving TURB, the level of CTCs identified in PV3 was significantly higher than that in PV1 (p = 0.047). However, there was no significant difference in CTC counts before and after resection in the RERBT group. Conclusion RERBT did not increase the number of tumor cells in the bloodstream.

Correction for ‘Fluorescent metabolic labeling-based quick antibiotic susceptibility test for anaerobic bacteria’ by Juan Gao et al., RSC Chem. Biol., 2022, https://doi.org/10.1039/d2cb00163b.

Specific modification of carbon dots (CDs) exhibit excellent properties and are suitable for numerous applications. The exploitation of multifunctional CDs in the area of fluorescent assay is still urgent and attractive. Herein, using a simple two-step heating procedure, positive charged carbon dots (P-CDs) were prepared with the co-doping of the elements of N and S. The morphology, optical properties and stability of P-CDs was investigated. Furthermore, P-CDs show the dual functions of unprecedented quenching performance toward fluorophore-modified DNA coupling the retaining fluorescence of P-CDs self. The quenching effect and mechanism of P-CDs to fluorophore-modified DNA was investigated. The fluorescence and quenching properties of P-CDs were used to design a ratiometric fluorescence platform for DNA based on the complex of P-CDs and FAM-labeled ssDNA. The ratiometric method allows the detection of target DNA because of the different affinities of P-CDs to ssDNA and dsDNA with acceptable sensitivity and improved reproducibility, when using sequence fragment of the gene of Gardnerella vaginalis as model. This method provides a new approach to widen the future of the biological applications based on some special CDs.

Pressure-based bioassays (PASS) integrate a molecular recognition process with a catalyzed gas generation reaction, enabling sensitive and portable quantitation of biomarkers in clinical samples. Using platinum nanoparticles (PtNPs) as a catalyst has significantly improved the sensitivity of PASS compared with protein enzyme-based detection. However, PtNPs are easily deactivated during storage or after being decorated with antibodies. Moreover, nonspecific adsorption of PtNPs on substrates has been a problem, resulting in significant backgrounds. To solve these problems of PtNP-based detection, we report a robust, simple, stable, and sensitive Pt staining method for PASS. Detection antibody-decorated gold nanoparticles (AuNPs) are used to perform enzyme-linked immunosorbent assay, followed by Pt staining to stain AuNPs with Ag and Pt bimetallic shells (Au@AgPtNPs), which endow AuNPs with catalytic activity. The concentration of targets can be quantitatively determined by measuring the pressure due to O2 g...

Digital microfluidics (DMF) is a powerful platform for a broad range of applications, especially immunoassays having multiple steps, due to the advantages of low reagent consumption and high automatization. Surface enhanced Raman scattering (SERS) has been proven as an attractive method for highly sensitive and multiplex detection, because of its remarkable signal amplification and excellent spatial resolution. Here we propose a SERS-based immunoassay with DMF for rapid, automated, and sensitive detection of disease biomarkers. SERS tags labeled with Raman reporter 4-mercaptobenzoic acid (4-MBA) were synthesized with a core@shell nanostructure and showed strong signals, good uniformity, and high stability. A sandwich immunoassay was designed, in which magnetic beads coated with antibodies were used as solid support to capture antigens from samples to form a beads-antibody-antigen immunocomplex. By labeling the immunocomplex with a detection antibody-functionalized SERS tag, antigen can be sensitively detected through the strong SERS signal. The automation capability of DMF can greatly simplify the assay procedure while reducing the risk of exposure to hazardous samples. Quantitative detection of avian influenza virus H5N1 in buffer and human serum was implemented to demonstrate the utility of the DMF-SERS method. The DMF-SERS method shows excellent sensitivity (LOD of 74 pg/mL) and selectivity for H5N1 detection with less assay time (1 h) and lower reagent consumption (∼30 μL) compared to the standard ELISA method. Therefore, this DMF-SERS method holds great potentials for automated and sensitive detection of a variety of infectious diseases.

As a rapidly developing field, paper-based microfluidic devices (μPADs) are expected to have profound implications, especially for in vitro diagnosis. In particular, integrated μPADs, with the merits of capillary-driven force, cost effectiveness, mass production and ease of use, are emerging as ideal tools for personal diagnosis; sample collection/pretreatment, signal transduction and results output can be simplified into one μPAD with minimal need for complex operation. In this review, we emphasize the latest developments of integrated μPADs and highlight the accomplishments and challenges of each component, including sample collection/pretreatment, signal transduction and amplification, and results output. Moreover, we give an overview of the growing trend of novel integrated μPADs and analyze existing challenges as well as potential directions for integrated μPADs for point-of-care testing (POCT).

Gas generation-based measurement is an attractive alternative approach for POC (Point-of-care) testing, which relies on the amount of generated gas to detect the corresponding target concentrations. In gas generation-based POC testing, the integration of a target recognition component and a catalyzed gas-generating reaction initiated by the target introduction can lead to greatly amplified signals, which can be highly sensitive measured via distance readout or simple hand-held devices. More importantly, numerous gas-generating reactions are environment-friendly since their products such as oxygen and nitrogen are nontoxic and odourless, which makes gas generation-based POC testing safe and secure for inexperienced staff. Researchers have demonstrated that gas generation-based measurements enable the rapid and highly sensitive POC detection of a variety of analytes. In this review, we focus on the recent developments in gas generation-based POC testing systems. The common types of gas-generating reactions are first listed and the translation of gas signals to different signal readouts for POC testing are then summarized, including distance readouts and hand-held devices. Moreover, we introduce gas bubbles as actuators to power microfluidic devices. We finally provide the applications and future perspective of gas generation-based POC testing systems.

The main function and biological processes of tissues are determined by the combination of gene expression and spatial organization of their cells. RNA sequencing technologies have primarily interrogated gene expression without preserving the native spatial context of cells. However, the emergence of various spatially-resolved transcriptome analysis methods now makes it possible to map the gene expression to specific coordinates within tissues, enabling transcriptional heterogeneity between different regions, and for the localization of specific transcripts and novel spatial markers to be revealed. Hence, spatially-resolved transcriptome analysis technologies have broad utility in research into human disease and developmental biology. Here, recent advances in spatially-resolved transcriptome analysis methods are summarized, including experimental technologies and computational methods. Strengths, challenges, and potential applications of those methods are highlighted, and perspectives in this field are provided.

Microfluidics have attracted increasing attention, as they can precisely manipulate the fluid in a microscale channel or structure. Microfluidics-based biochemical sensing has been popularly used for various applications benefitting from the intrinsic advantages of low reagent consumption, rapid reaction rate, high throughput, easy integration, and facile automation. However, microfluidic device-based biochemical sensing is still limited by conventional signal readout that relies on sophisticated instruments and trained operators. Hence, the exploration of simplified signal readout is a critical issue in the development of microfluidic device-based biochemical sensing. In this review, equipment-free and low-cost signaling approaches for microfluidics are summarized, including colorimetric-readout, distance-readout, and smartphone-readout. Several other simplified signal readouts with potential to be integrated into microfluidics are described as well. Additionally, the prospects and challenges of simplified signal readout-based microfluidic devices are discussed.

Since the transferrin receptor (CD71 or TFRC) is known to be highly expressed in numerous cancers, CD71 has become an attractive target in cancer research. Acquiring specific molecular probes for CD71, such as small molecular ligands, aptamers, peptides, or antibodies, is of great importance for cancer cell recognition and capture. In this work, we chose CD71 as the target for phage display, and after four rounds of positive selection and one round of negative selection, the specific phage library was enriched. After verification and sequence analysis, six peptides were identified to be able to bind to CD71 with high specificity. The specific recognition of the CD71-positive cells was confirmed by flow cytometry and confocal microscopy. Competition experiments demonstrated that peptide Y1 and transferrin (TF) were bound to distinct sites on CD71, indicating that peptide Y1 could replace TF as a potential probe for cell imaging and drug delivery, thus avoiding competition by endogenous TF and side effects. Graphical abstract Six peptides were successfully isolated using in vitro biopanning against CD71 with high specificity and affinity. Peptides Y1 and Y2 would be powerful tools in biosensors and biomedicine due to their unique properties.

Enzyme-linked immunosorbent assay (ELISA) is a popular laboratory technique for detection of disease-specific protein biomarkers with high specificity and sensitivity. However, ELISA requires labor-intensive and time-consuming procedures with skilled operators and spectroscopic instrumentation. Simplification of the procedures and miniaturization of the devices are crucial for ELISA-based point-of-care (POC) testing in resource-limited settings. Here, we present a fully integrated, instrument-free, low-cost and portable POC platform which integrates the process of ELISA and the distance readout into a single microfluidic chip. Based on manipulation using a permanent magnet, the process is initiated by moving magnetic beads with capture antibody through different aqueous phases containing ELISA reagents to form bead/antibody/antigen/antibody sandwich structure, and finally converts the molecular recognition signal into a highly sensitive distance readout for visual quantitative bioanalysis. Without additional equipment and complicated operations, our integrated ELISA-Chip with distance readout allows ultrasensitive quantitation of disease biomarkers within 2h. The ELISA-Chip method also showed high specificity, good precision and great accuracy. Furthermore, the ELISA-Chip system is highly applicable as a sandwich-based platform for the detection of a variety of protein biomarkers. With the advantages of visual analysis, easy operation, high sensitivity, and low cost, the integrated sample-in-answer-out ELISA-Chip with distance readout shows great potential for quantitative POCT in resource-limited settings.

This review aims to provide in-depth insights into CTC analysis, including various techniques for isolation of CTCs and single-cell analysis of CTCs, as well as current developmental trends and promising research directions., Up to 90% of cancer-related deaths are caused by metastatic cancer. Circulating tumor cells (CTCs), a type of cancer cell that spreads through the blood after detaching from a solid tumor, are essential for the establishment of distant metastasis for a given cancer. As a new type of liquid biopsy, analysis of CTCs offers the possibility to avoid invasive tissue biopsy procedures with practical implications for diagnostics. The fundamental challenges of analyzing and profiling CTCs are the extremely low abundances of CTCs in the blood and the intrinsic heterogeneity of CTCs. Various technologies have been proposed for the enrichment and single-cell analysis of CTCs. This review aims to provide in-depth insights into CTC analysis, including various techniques for isolation of CTCs with capture methods based on physical and biochemical principles, and single-cell analysis of CTCs at the genomic, proteomic and phenotypic level, as well as current developmental trends and promising research directions.

This paper describes a centrifugal micropipette-tip method for ELISA sample processing combined with a pressure meter for portable quantitative detection of acute myocardial infarction (AMI) biomarker myoglobin (Myo). The method enables sensitive and reliable quantification of Myo in 35 minutes. With the advantages of simplicity, speed, user friendliness, low cost, and small sample consumption, centrifugal micropipette-tip ELISA shows great potential for quantitative POC diagnostics for AMI.

Molecular recognition ligands are of great significance in many fields, but our ability to develop new recognition molecules remains to be expanded. Here, we developed a Sequential Multidimensional Analysis algoRiThm for aptamer discovery (SMART-Aptamer) from high-throughput sequencing (HTS) data of SELEX libraries based on multilevel structure analysis and unsupervised machine learning to discover nucleic acid recognition ligands with high accuracy and efficiency. We validated SMART-Aptamer with three sets of HTS data from screening pools against hESCs, EpCAM, and CSV. High affinity aptamers for all three targets were successfully obtained, and the results revealed that SMART-Aptamer is able to pick out high affinity aptamers with low false positive and negative rates. With the advantages of accuracy, efficiency, and robustness, SMART-Aptamer represents a paradigm-shift strategy for the discovery of binding ligands for a variety of biomedical applications.

An enthalpy-driven ligand is an ideal probe for practical applications because of the formation of abundant specific bonds between the ligand and target, compared to an entropy-driven ligand with a similar Gibbs free energy change. However, there has been a lack of direct discovery strategy for identifying enthalpy-driven ligands. In this work, a molecular crowding SELEX (systematic evolution of ligands by exponential enrichment) strategy for discovering enthalpy-driven aptamers was developed to improve the affinity and selectivity of aptamers in complex samples. Three aptamer sequences were successfully evolved against a tumor biomarker protein, and all proved to be enthalpy-driven by thermodynamics analysis, establishing the feasibility of molecular crowding SELEX for effective discovery of enthalpy-driven aptamers. Further comparison of aptamers evolved from conventional SELEX in buffer and molecular crowding SELEX (SYL-H2C) revealed much higher affinity of SYL-H2C. With its improved thermodynamic properties, the enthalpy-driven SYL-H2C aptamer was able to detect circulating tumor cells in real cancer patient blood samples with excellent detection accuracy (10/10). The proposed molecular crowding screening strategy offers a promising direction for discovering robust binding probes for a great variety of biomedical applications.

As the basic units of life, cells present dramatic heterogeneity which, although crucial to an organism's behavior, is undetected by bulk analysis. Recently, much attention has been paid to reveal cellular types and states at the single-cell level including genome, transcriptome, epigenome or proteome-based on sequencing and immunological methods, etc. Among these approaches, transcriptomic analysis provides knowledge of the molecular linkages between genetic information and the proteome, leading to a comprehensive understanding of biological processes and diseases. Compared to single-dimensional inspection, multi-dimensional analysis combines the transcriptome with other "omics" to enable a comprehensive understanding of single-cell processes and functions. Moreover, compared to separate observations or single snapshots, spatial dimension and lineage time tracing can provide a more multifaceted understanding of the micro-environment and dynamic processes. In this review, we will introduce current transcriptomic analysis methods, as well as their combination with other omics methods including genomic, proteomic, and epigenetic approaches. Multi-dimensional analysis using these approaches for spatial positioning and lineage tracing applications will be reviewed. The future perspectives of single-cell multi-omics analysis based on the transcriptome will also be discussed.

Functionalization of gold nanoparticles (AuNPs) with biomolecules such as DNA is an important step to provide recognition capability for applications in bioanalysis. This chapter summarizes the methods for synthesizing AuNPs, and especially for functionalizing AuNPs with DNA including the traditional salt-aging method; the recently developed methods, such as the approaches assisted by sonication, low pH, or freezing; and the synergistically assisted methods.The principles, advantages, and disadvantages of each approach are discussed. Finally, the application in the bioanalysis of DNA-conjugated AuNPs is reviewed.

Cells are the basic building blocks of biological systems, with inherent unique molecular features and development trajectories. The study of single cells facilitates in-depth understanding of cellular diversity, disease processes, and organization of multicellular organisms. Single-cell RNA sequencing (scRNA-seq) technologies have become essential tools for the interrogation of gene expression patterns and the dynamics of single cells, allowing cellular heterogeneity to be dissected at unprecedented resolution. Nevertheless, measuring at only transcriptome level or 1D is incomplete; the cellular heterogeneity reflects in multiple dimensions, including the genome, epigenome, transcriptome, spatial, and even temporal dimensions. Hence, integrative single cell analysis is highly desired. In addition, the way to interpret sequencing data by virtue of bioinformatic tools also exerts critical roles in revealing differential gene expression. Here, a comprehensive review that summarizes the cutting-edge single-cell transcriptome sequencing methodologies, including scRNA-seq, spatial and temporal transcriptome profiling, multi-omics sequencing and computational methods developed for scRNA-seq data analysis is provided. Finally, the challenges and perspectives of this field are discussed.

An aptamer blocking strategy is developed by Honglin Chen, Yanling Song, Chaoyong Yang, and co‐workers in their Research Article on page 10266 to inhibit SARS‐CoV‐2 infection. An aptamer probe was engineered to bind to the spike protein of SARS‐CoV‐2, which inhibits infection with the virus by blocking the interaction of the virus with ACE2 receptors on the human cell membrane.

Lanthanide elements (Ln) play an important role in industry and agriculture. As a result of the increasing consumption of lanthanides, environmental emission of Ln has become detrimental to the health of flora and fauna. Current methods for trace lanthanides detection mainly rely on sophisticated instruments. In this article, a Ln3+ dependent DNAzyme was incorporated into a hydrogel to generate Ln3+ sensitive DNAzyme hydrogel for portable colorimetric detection. The enzyme strand and its substrate strand act as crosslinker and functional unit of the hydrogel with polyacrylamide chains as the scaffold and gold nanoparticles (AuNPs) as the indicator of hydrogel stability. Any ions in the Ln3+ series can trigger the cleavage of substrate strand by activating the enzyme strand, thereby decreasing the crosslink ratio and leading to collapse of the hydrogel. The release of the encapsulated AuNPs turns the supernatant wine red. Using this colorimetric method, Ln3+ can be detected with high sensitivity, with a limit of detection (LOD) of 20 nM for Ce3+. The hydrogel responds specifically to any Ln3+ ion and works well with the spiked lake sample without the need of instruments and skilled operators. Our results suggest that the lanthanide responsive hydrogel can be used for portable and sensitive detection of Ln3+ contamination in the field.

It is demonstrated that DNA can be used to control the synthesis of silver nanoplates with different morphologies using spherical silver seeds. UV-vis spectroscopy, transmission electron microscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, and Raman spectroscopy are used to characterize the synthesized nanoparticles. Silver nanoprisms are encoded by poly C and poly G, while silver flower bouquets and silver nanodiscs are synthesized using poly A and poly T, respectively. The length of DNA is found to have little effect on the morphology of silver nanoparticles. Moreover, the synthesized silver nanoplates are found to have high surface enhanced Raman scattering enhancement ability, good antibacterial activity, and good biocompatibility. These discoveries will broaden the application of DNA in nanoscience and will provide a new platform to investigate the interaction between DNA sequences and silver nanoparticles.

Due to its large enhancement effect, nanostructure-based surface-enhanced Raman scattering (SERS) technology had been widely applied for bioanalysis and cell imaging. However, most SERS nanostructures suffer from poor signal reproducibility, which hinders the application of SERS nanostructures in quantitative detection. We report an etching-assisted approach to synthesize SERS-active plasmonic nanoparticles with 1 nm interior nanogap for multiplex quantitative detection and cancer cell imaging. Raman dyes and methoxy poly(ethylene glycol) thiol (mPEG-SH) were attached to gold nanoparticles (AuNPs) to prepare gold cores. Next, Ag atoms were deposited on gold cores in the presence of Pluronic F127 to form a Ag shell. HAuCl4 was used to etch the Ag shell and form an interior nanogap in Au@AgAuNPs, leading to increased Raman intensity of dyes. SERS intensity distribution of Au@AgAuNPs was found to be more uniform than that of aggregated AuNPs. Finally, Au@AgAuNPs were used for multiplex quantitative detection and cancer cell imaging. With the advantages of simple and rapid preparation of Au@AgAuNPs with highly uniform, stable, and reproducible Raman intensity, the method reported here will widen the applications of SERS-active nanoparticles in diagnostics and imaging.