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Highlights A simple and convenient graphene bio-interface was designed by using multifunctional nano-denatured bovine serum albumin (nano-dBSA) film. Highly sensitive cancer biomarker detection in diluted serum at the femtogram per milliliter level was achieved using the nano-dBSA functionalized graphene field-effect transistor. Electronic supplementary material The online version of this article (10.1007/s40820-019-0250-8) contains supplementary material, which is available to authorized users., A simple, convenient, and highly sensitive bio-interface for graphene field-effect transistors (GFETs) based on multifunctional nano-denatured bovine serum albumin (nano-dBSA) functionalization was developed to target cancer biomarkers. The novel graphene–protein bioelectronic interface was constructed by heating to denature native BSA on the graphene substrate surface. The formed nano-dBSA film served as the cross-linker to immobilize monoclonal antibody against carcinoembryonic antigen (anti-CEA mAb) on the graphene channel activated by EDC and Sulfo-NHS. The nano-dBSA film worked as a self-protecting layer of graphene to prevent surface contamination by lithographic processing. The improved GFET biosensor exhibited good specificity and high sensitivity toward the target at an ultralow concentration of 337.58 fg mL−1. The electrical detection of the binding of CEA followed the Hill model for ligand–receptor interaction, indicating the negative binding cooperativity between CEA and anti-CEA mAb with a dissociation constant of 6.82 × 10−10 M. The multifunctional nano-dBSA functionalization can confer a new function to graphene-like 2D nanomaterials and provide a promising bio-functionalization method for clinical application in biosensing, nanomedicine, and drug delivery. Electronic supplementary material The online version of this article (10.1007/s40820-019-0250-8) contains supplementary material, which is available to authorized users.

Microspheres coated with specific biomolecules have been broadly employed to detect tumor markers. In this study, we developed a novel microfluidic platform based on pressure difference for the uniform distribution of microspheres and applied this platform for carcinoma-embryonic antigen detection with quantum dots probes. Within our microfluidic device, micropost arrays were designed to ensure that the trapped microspheres formed dot arrays to overcome the shortcomings of clogging and loss of microspheres, which typically occurred in currently used structures for microsphere capture. A sandwich immunoassay for the detection of typical cancer biomarker CEA was successfully performed on the microfluidic device with all reagents reacted together, which led to reduced experimental time and simple operations. As a result, a limit of detection of 0.5 ng/mL for carcinoma-embryonic antigen detection was achieved, and the calibration curve fitted the Langmuir model, which showed high clinical consistency with serum sample detection. To further investigate the performance of our system, trap efficiency and uniform ratio of microfluidic structure, and specificity of the experiment were evaluated and showed good consequences. The proposed microfluidic immunoassay system with advantages of high sensitivity and specificity, reduced time cost, and facile operations, indicates a promising potential in clinical diagnosis.

High sensitive digital polymerase chain reaction (PCR) is one of the novel technology in molecular biology and has become more and more important in the arena of nucleic acid quantification. However, existing digital PCR technologies usually require specialized platform like high sensitive fluorescence microscope or flow cytometer, which has increased the detection costs and limited their further applications. Here we developed a simple and low cost nucleic acid quantification method based on beads, emulsion, amplification, and magnetics (BEAMing), microsphere and microcolumn array chip. We used the streptavidin (SA) modified polystyrene microspheres to capture the biotin covered target beads obtained via BEAMing technology, which could represent the population of target DNA. The microspheres-beads complexes were then collected and trapped in a microcolumn array chip and observed under a normal microscope. Because the number of microspheres-beads complexes had a relationship of coupled Poisson distribution with the number of target DNA, we could easily infer the number of target DNA through counting the number of chip-trapped microspheres. Our method has reduced the costs and lowers down the difficulty of digital PCR technology, which may promote the applications of digital PCR in research and clinical field.

In this work, a real-time assay for highly sensitive, label-free, multiplexed electrical detection of lung cancer biomarkers was developed by using silicon nanowire field-effect (SiNW-FET) devices. Highly responsive SiNW arrays were fabricated using a CMOS-compatible anisotropic self-stop etching technique with mass reproducibility and low cost character. The SiNW nanosensor was integrated with PDMS microfluidic device, which allows rapid analyte delivery, makes the analysis to be conducted using exceedingly small samples and enables potential multiplexed detection. The nanowire arrays allowed highly selective and sensitive multiplexed detection of microRNA (miRNA)−126 and CEA. Due to high surface-to-volume ratio that the nanowire dimensions confer, the detection floor of single molecule was achieved. The potential utility in identifying clinical samples for early diagnosis of cancer was demonstrated by analyzing biomarkers in clinical related samples. The developed nanosensor with capability for multiplexed real-time monitoring of biomarkers with high sensitivity and selectivity in clinically relevant samples is highly attractive for diagnosis and treatment of cancer and other diseases.

We propose the first black phosphorus (BP) – fiber optic biosensor for ultrasensitive diagnosis of human neuron-specific enolase (NSE) cancer biomarkers. A novel optical-nano configuration has been exploited by integrating BP nanosheets with a largely tilted fiber grating (BP-TFG), where the BP is bio-functionalized by the poly-L-lysine acting as a critical cross-linker to facilitate bio-nano-photonic interface with extremely enhanced light-matter interaction. BP nanosheets are synthesized by a liquid ultrasonication-based exfoliation and deposited on fiber device by an in-situ layer-by-layer method. The BP-induced optical modulation effects in terms of thickness-tunable feature, polarization-dependence and enhanced light-matter interaction are experimentally investigated. The anti-NSE immobilized BP-TFG biosensor has been implemented to detect NSE biomarkers demonstrating ultrahigh sensitivity with limit of detection down to 1.0 pg/mL, which is 4 orders magnitude lower than NSE cut-off value of small cell lung cancer. The enhanced sensitivity of BP-TFG is 100-fold higher than graphene oxide or AuNPs based biosensors. We believe that BP-fiber optic configuration opens a new bio-nano-photonic platform for the applications in healthcare, biomedical, food safety and environmental monitoring.

In recent years, nanopore technology has become increasingly important in the field of life science and biomedical research. By embedding a nano-scale hole in a thin membrane and measuring the electrochemical signal, nanopore technology can be used to investigate the nucleic acids and other biomacromolecules. One of the most successful applications of nanopore technology, the Oxford Nanopore Technology, marks the beginning of the fourth generation of gene sequencing technology. In this review, the operational principle and the technology for signal processing of the nanopore gene sequencing are documented. Moreover, this review focuses on the applications using nanopore gene sequencing technology, including the diagnosis of cancer, detection of viruses and other microbes, and the assembly of genomes. These applications show that nanopore technology is promising in the field of biological and biomedical sensing.

Nanotechnology has found wide use in biomedical applications and the food and bioprocessing industry. In this light, we demonstrate a facile strategy to prepare bifunctional monodisperse silica nanospheres encapsulating chitosan-coated magnetic nanoparticles and CdTe quantum dots. The size of these composite spheres can be adjusted from 90 nm to 500 nm by varying the concentration of ammonia, water, tetraethyl orthosilicate, and the ratio of the chitosan-coated magnetic nanoparticles and CdTe quantum dots. The composite spheres are characterized using scanning electron microscope analyses, transmission electron microscope analyses, energy-dispersed spectrum studies, Malvern Zetasizer, vibrating sample magnetometer, and fluorescence microscopy. The spheres exhibit good monodispersion and favorable superparamagnetic and fluorescent properties. The luminescence of the spheres can be varied by using different types of coated quantum dots. Such composite spheres with tunable characteristics allow for external manipulation of research systems by magnetic fields together with the real-time fluorescent monitoring of multiple samples. The abovementioned properties can potentially be exploited for application in the biomedical and biosensing fields.

BEAMing (beads, emulsion, amplification, and magnetics) is a high sensitive emulsion digital PCR (polymerase chain reaction) technology. Target beads obtained via BEAMing can be used to determine the copy number of target DNA population. However, the existing beads counting process requires specialized instrument, which has increased the detection costs. Here we developed an easy and low cost beads counting method combined with microspheres and micropillar array chips. Through counting the chip-trapped polystyrene microspheres which have captured the target magnetic beads, we could easily infer the number of target magnetic beads and the copy number of target DNA. The trapping mechanism was based on the difference of microsphere diameter and pillar intervals. Fabrication of the chip was simple and low-cost. This method has reduced the difficulty of beads counting process in BEAMing and may promote the applications of BEAMing in clinical filed.

Developing a rapid, accurate and sensitive electrochemical biosensor for detecting cancer biomarkers is important for early detection and diagnosis. This work reports an electrochemical biosensor based on a graphene (GR) platform which is made by CVD, combined with magnetic beads (MBs) and enzyme-labeled antibody-gold nanoparticle bioconjugate. MBs coated with capture antibodies (Ab1) were attached to GR sheets by an external magnetic field, to avoid reducing the conductivity of graphene. Sensitivity was also enhanced by modifying the gold nanoparticles (AuNPs) with horseradish peroxidase (HRP) and the detection antibody (Ab2), to form the conjugate Ab2–AuNPs–HRP. Electron transport between the electrode and analyte target was accelerated by the multi-nanomaterial, and the limit of detection (LOD) for carcinoembryonic antigen (CEA) reached 5 ng mL−1. The multi-nanomaterial electrode GR/MBs–Ab1/CEA/Ab2–AuNPs–HRP can be used to detect biomolecules such as CEA. The EC biosensor is sensitive and specific, and has potential in the detection of disease markers.

In recent years, quantum dots (QDs) have been widely used in numerous biological processes and bio-analysis as novel fluorescence indicators. In the study, we developed a quantum dot-based bio-barcode amplification in combination with a Taq DNA ligase approach for the rapid detection of a polymorphism in kinesin-like protein 6 gene (KIF6), which significantly enhances the sensitivity. A ligand-exchange reaction was performed to prepare MPA-functionalized water-soluble QDs. The amine-modified oligonucleotides were coupled to QDs surface to form functional QDs-DNA conjugates. Meanwhile, the surface of gold nanoparticles (AuNPs) was modified with both the detection probe and bio-barcode oligonucleotides. Then, the amine-modified capture DNAs were immobilized onto the chip, followed by the addition of the modified AuNPs to the modified chip surface to form sandwiched hybridization. When the capture DNA and detection probe were perfectly matched to target DNA (KIF6), Taq DNA ligase in the solution would link t...

Two-dimensional graphene devices are widely used for biomolecule detection. Nevertheless, the surface modification of graphene is critical to achieve the high sensitivity and specificity required for biological detection. Herein, native bovine serum albumin (BSA) in inorganic solution is denatured on the graphene surface by heating, leading to the formation of nanoscale BSA protein films adsorbed on the graphene substrate via π-stacking interactions. This technique yields a controllable, scalable, uniform, and high-coverage method for graphene biosensors. Further, the application of such nanoscale heat-denatured BSA films on graphene as a universal graphene biosensor platform is explored. The thickness of heat-denatured BSA films increased with heating time and BSA concentration but decreased with solvent concentration as confirmed by atomic force microscopy. The noncovalent interaction between denatured BSA films and graphene was investigated by Raman spectroscopy. BSA can act as a p-type and n-type dopant by modulating pH-dependent net charges on the layered BSA-graphene surface, as assessed by current-voltage measurements. Chemical groups of denatured BSA films, including amino and carboxyl groups, were verified by X-ray photoelectron microscopy, attenuated total reflectance-Fourier transform infrared spectra, and fluorescent labeling. The tailoring of the BSA-graphene surfaces through chemical modification, controlled thickness, and doping type via noncovalent interactions provides a controllable, multifunctional biosensor platform for molecular diagnosis without the possibility of nonspecific adsorption on graphene.

In this study, arrays of controllable, Ge-SiO2 core-shell structure nanowire (NW) were fabricated by combining the complementary metal-oxide semiconductor (CMOS) compatible technology and Ge-condensation technology. Meanwhile, based on this novel nanostructure, the biosensor application in label-free, highly sensitive detection of lung cancer biomarker was investigated. This nanosensor illuminated highly sensitive concentration dependent conductance change in response to targets and had high specificity in one-base mismatched target DNA discrimination.

We report here an optical approach that enables highly selective and colorimetric single-base mismatch detection without the need of target modification, precise temperature control or stringent washes. The method is based on the finding that nucleoside monophosphates (dNMPs), which are digested elements of DNA, can better stabilize unmodified gold nanoparticles (AuNPs) than single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) with the same base-composition and concentration. The method combines the exceptional mismatch discrimination capability of the structure-selective nucleases with the attractive optical property of AuNPs. Taking S1 nuclease as one example, the perfectly matched 16-base synthetic DNA target was distinctively differentiated from those with single-base mutation located at any position of the 16-base synthetic target. Single-base mutations present in targets with varied length up to 80-base, located either in the middle or near to the end of the targets, were all effectively detected. In order to prove that the method can be potentially used for real clinic samples, the single-base mismatch detections with two HBV genomic DNA samples were conducted. To further prove the generality of this method and potentially overcome the limitation on the detectable lengths of the targets of the S1 nuclease-based method, we also demonstrated the use of a duplex-specific nuclease (DSN) for color reversed single-base mismatch detection. The main limitation of the demonstrated methods is that it is limited to detect mutations in purified ssDNA targets. However, the method coupled with various convenient ssDNA generation and purification techniques, has the potential to be used for the future development of detector-free testing kits in single nucleotide polymorphism screenings for disease diagnostics and treatments.

We report here a quantum dots-DNA (QDs-DNA) nanosensor based on fluorescence resonance energy transfer (FRET) for the detection of the target DNA and single mismatch in hepatitis B virus (HBV) gene. The proposed one-pot DNA detection method is simple, rapid and efficient due to the elimination of the washing and separation steps. In this study, the water-soluble CdSe/ZnS QDs were prepared by replacing the trioctylphosphine oxide (TOPO) on the surface of QDs with 3-mercaptopropionic acid (MPA). Subsequently, oligonucleotides were attached to the QDs surface to form functional QDs-DNA conjugates. Along with the addition of DNA targets and Cy5-modified signal DNAs into the QDs-DNA conjugates, sandwiched hybrids were formed. The resulting assembly brings the Cy5 fluorophore, the acceptor, and the QDs, the donor, into proximity, leading to fluorescence emission from the acceptor by means of FRET on illumination of the donor. In order to efficiently detect single-base mutants in HBV gene, oligonucleotide ligation assay was employed. If there existed a single-base mismatch, which could be recognized by the ligase, the detection probe was not ligated and no Cy5 emission was produced due to the lack of FRET. The feasibility of the proposed method was also demonstrated in the detection of synthetic 30-mer oliginucleotide targets derived from the HBV with a sensitivity of 4.0 nM by using a multilabel counter. The method enables a simple and efficient detection that could be potentially used for high throughput and multiplex detections of target DNA and the mutants.

A direct, rapid, highly sensitive and specific biosensor for detection of cancer biomarkers is desirable in early diagnosis and prognosis of cancer. However, the existing methods of detecting cancer biomarkers suffer from poor sensitivity as well as the requirement of enzymatic labeling or nanoparticle conjugations. Here, we proposed a two-channel PDMS microfluidic integrated CMOS-compatible silicon nanowire (SiNW) field-effect transistor arrays with potentially single use for label-free and ultrasensitive electrical detection of cancer biomarkers. The integrated nanowire arrays showed not only ultrahigh sensitivity of cytokeratin 19 fragment (CYFRA21-1) and prostate specific antigen (PSA) with detection to at least 1 fg/mL in buffer solution but also highly selectivity of discrimination from other similar cancer biomarkers. In addition, this method was used to detect both CYFRA21-1 and PSA real samples as low as 10 fg/mL in undiluted human serums. With its excellent properties and miniaturization, the integrated SiNW-FET device opens up great opportunities for a point-of-care test (POCT) for quick screening and early diagnosis of cancer and other complex diseases.

Isolation of circulating tumor cells (CTCs) by size exclusion is a widely researched technique that offers the advantage of capturing tumor cells without reliance on cell surface expression markers. In this work, we report the development of a novel polydimethylsiloxane (PDMS) membrane filter-based microdevice for rapid and highly efficient isolation of CTCs from peripheral blood. A precise and highly porous PDMS microfilter was fabricated and integrated into the microfiltration chip by combining a sacrificial transferring film with a sandwich molding method. We achieved >90% recovery when isolating lung cancer cells from spiked blood samples, with a relatively high processing throughput of 10 mL/h. In contrast to existing CTC filtration systems, which rely on low-porosity track-etch filters or expensive lithography-based filters, our microfiltration chip does not require complex e-beam lithography or the reactive ion etching process, therefore it offers a low-cost alternative tool for highly efficient CTC enrichment and in situ analysis. Thus, this new microdevice has the potential for use in routine monitoring of cancer development and cancer therapy in a clinical setting.

Herein, we describe a novel approach for rapid, label-free and specific DNA detection by applying rolling circle amplification (RCA) based on silicon nanowire field-effect transistor (SiNW-FET) for the first time. Highly responsive SiNWs were fabricated with a complementary metal oxide semiconductor (CMOS) compatible anisotropic self-stop etching technique which eliminated the need for hybrid method. The probe DNA was immobilized on the surface of SiNW, followed by sandwich hybridization with the perfectly matched target DNA and RCA primer that acted as a primer to hybridize the RCA template. The RCA reaction created a long single-stranded DNA (ssDNA) product and thus enhanced the electronic responses of SiNW significantly. The signal-to-noise ratio (SNR) as a figure-of-merit was analyzed to estimate the signal enhancement and possible detection limit. The nanosensor showed highly sensitive concentration-dependent conductance change in response to specific target DNA sequences. Because of the binding of an abundance of repeated sequences of RCA products, the SNR of20 for 1 fM DNA detection was achieved, implying a detection floor of 50 aM. This RCA-based SiNW biosensor also discriminated perfectly matched target DNA from one-base mismatched DNA with high selectivity due to the substantially reduced nonspecific binding onto the SiNW surface through RCA. The combination of SiNW FET sensor with RCA will increase diagnostic capacity and the ability of laboratories to detect unexpected viruses, making it a potential tool for early diagnosis of gene-related diseases.

Electrochemical detection strategies employing nanotechnologies which include a variety of new materials and fabrication processes offer new opportunities for highly sensitive detection of cancer biomarkers. This work reports an electrochemical biosensor based on a graphene (GR) platform, combined with magnetic beads (MBs) and enzyme-labeled antibody-gold nanoparticle bioconjugates. MBs coated with capture antibodies (Ab1) were attached to isolated GR sheets by an external magnetic field, to avoid reducing the conductivity of graphene. Sensitivity was also enhanced by modifying the gold nanoparticles (AuNPs) with horseradish peroxidase (HRP) and the detection antibody (Ab2), to form the conjugate Ab2-AuNPs-HRP. Electron transport between the electrode and analyte target was accelerated by the multi-nanomaterial, and the limit of detection (LOD) for carcinoembryonic antigen (CEA) reached 5ng/mL, which meets clinical requirements. The multi-nanomaterial electrode GR/MBs-Ab1/Antigen/Ab2-AuNPs-HRP can be used to detect biomolecules such as CEA. The EC biosensor is sensitive and specific, and has potential in the detection of disease markers.Keywords: electrochemical, graphene,magnetic beads, gold nanoparticles, horseradish peroxidase.View Large Image | View Hi-Res Image | Download PowerPoint Slide

This paper reports the design and implementation of a contactless conductivity detection system which combines a thermal control cell, a data processing system and an electrochemical (EC) cell for label-free isothermal nucleic acid amplification and real-time monitoring. The EC cell consists of a microchamber and interdigitated electrodes as the contactless conductivity biosensor with a cover slip as insulation. In our work, contactless EC measurements, the effects of trehalose on amplification, and chip surface treatment are investigated. With the superior performance of the biosensor, the device can detect the amount of pure DNA at concentrations less than 0.1 pg μl(-1). The EC cell, integrated with a heater and a temperature sensor, has successfully implemented nicking-based strand-displacement amplification at an initial concentration of 2.5 μM and the yields are monitored directly (dismissing the use of probes or labels) on-line. This contactless detector carries important advantages: high anti-interference capability, long detector life, high reusability and low cost. In addition, the small size, low power consumption and portability of the detection cell give the system the potential to be highly integrated for use in field service and point of care applications.