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MicroRNAs (miRNAs), as the small, non-coding, evolutionary conserved, and post-transcriptional gene regulators of the genome, have been highly associated with various diseases such as cancers, viral infections, and cardiovascular diseases. Several techniques have been established to detect miRNAs, including northern blotting, real-time polymerase chain reaction (RT-PCR), and fluorescent microarray platform. However, it remains a significant challenge to develop sensitive, accurate, rapid, and cost-effective methods to detect miRNAs due to their short size, high similarity, and low abundance. The electrochemical biosensors exhibit tremendous potential in miRNA detection because they satisfy feature integration, portability, mass production, short response time, and minimal sample consumption. This article reviewed the working principles and signal amplification strategies of electrochemical DNA biosensors summarized the recent improvements. With the development of DNA nanotechnology, nanomaterials and biotechnology, electrochemical DNA biosensors of high sensitivity and specificity for microRNA detection will shortly be commercially accessible.

While doxorubicin (DOX) is one of the most common chemotherapeutic drugs for treating cancer, use of DOX must be managed carefully due to dose-related toxicity. Nanodiamond (ND) drug delivery system conjugated with DOX (NDX) has been reported to enhance treatment efficacy and attenuate toxicity in murine cancer models. In addition, extensive biocompatibility studies indicate that NDs seem to be well tolerated in non-human primates. Before the clinical translation of NDX, it is necessary to verify the safety of ND in large mammals. Studies of nanomedicine drug safety for large animal are not commonly reported, and this work represents a key milestone in bridging earlier advances towards clinical assessment. Herein, NDs’ safety as a drug-delivery platform was evaluated in Naive Beagle dogs. The study is performed with DOX, ND, and NDX in a dual-gender animal model using intravenous (IV) injection and hepatic portal vein (HPV) injection methods. The dogs are monitored for their health phenotype changes in continuous 5 days. Blood and urine obtained are for clinical pathology research. The results indicate that ND drug delivery platform significantly relieves DOX toxicity for Naive Beagle dog model. This study provides guidance for the pre-clinical safety assessment of NDX therapy at large animal level.

Protein coating is a strategy for modifying and improving the surface functional properties of nanomaterials. However, the underlying mechanism behind protein coating formation, which is essential for its practical applications, remains largely unknown. Herein, we investigate the fundamental molecular mechanism of protein coating formation. Polydopamine nanospheres (PDANS) coated with bovine serum albumin (BSA) are examined in this study due to their wide biomedical potential. Our results demonstrate that BSAs can flexibly bind to PDANS and maintain their structural dynamicity. Our findings unveil that regular structure formation arises from BSAs lateral interactions via electrostatic forces. Notably, the protein coating modified PDANS surface enhances cell adhesion and proliferation as well as osteogenic differentiation. Such an enhancement is attributed to complementary surface properties provided by the dynamic PDANS-BSA complex and regular structure caused by BSA-BSA interactions in protein coating formation. This study provides a fundamental understanding of the molecular mechanism of protein coating formation, which facilitates the further development of functional protein-coated nanomaterials and guides the bioengineering decision making for biomedical applications, especially in bone tissue engineering.

Typography-like templates for polydimethylsiloxane (PDMS) microfluidic chips using a fused deposition modeling (FDM) three-dimensional (3D) printer are presented. This rapid and fast proposed scheme did not require complicated photolithographic fabrication facilities and could deliver resolutions of ~100 μm. Polylactic acid (PLA) was adopted as the material to generate the 3D-printed units, which were then carefully assembled on a glass substrate using a heat-melt-curd strategy. This craft of bonding offers a cost-effective way to design and modify the templates of microfluidic channels, thus reducing the processing time of microfluidic chips. Finally, a flexible microfluidic chip to be employed for cell-based drug screening was developed based on the modularized 3D-printed templates. The lithography-free, typography-like, 3D-printed templates create a modularized fabrication process and promote the prevalence of integrated microfluidic systems with minimal requirements and improved efficiency.

Pancreatic cancer, at unresectable advanced stages, presents poor prognoses, which could be prevented by early pancreatic cancer diagnosis methods. Recently, a promising early-stage pancreatic cancer biomarker, extracellular vesicles (EVs) related glypican-1 (GPC1) mRNA, is found to overexpress in pancreatic cancer cells. Current mRNA detection methods usually require expensive machinery, strict preservation environments, and time-consuming processes to guarantee detection sensitivity, specificity, and stability. Herein, we propose a novel two-step amplification method (CHAGE) via the target triggered Catalytic Hairpin Assembly strategy combined with Gold-Enhanced point-of-care-testing (POCT) technology for sensitive visual detection of pancreatic cancer biomarker. First, utilizing the catalyzed hairpin DNA circuit, low expression of the GPC1 mRNA was changed into amplification product 1 (AP1, a DNA duplex) as the next detection targets of the paper strips. Second, the AP1 was loaded onto a lateral flow assay and captured with the gold signal nanoparticles to visualize results. Finally, the detected results can be further enhanced by depositing gold to re-enlarge the sizes of gold nanoparticles in detection zones. As a result, the CHAGE methodology lowers the detection limit of mRNA to 100 fM and provides results within 2 h at 37 °C. Furthermore, we demonstrate the successful application in discriminating pancreatic cancer cells by analyzing EVs’ GPC1 mRNA expression levels. Hence, the CHAGE methodology proposed here provides a rapid and convenient POCT platform for sensitive detection of mRNAs through unique probes designs (COVID, HPV, etc.). Supplementary Information The online version contains supplementary material available at 10.1186/s12951-021-01039-4.

The boosting exploitation of graphene oxide (GO) increases exposure risk to human beings. However, as the first line defender, neutrophils’ mechanism of defensive behavior towards GO invasion remains unclear. Herein, we discover that neutrophils defensively degrade GO in a lateral dimension dependent manner. The micrometer-sized GO (mGO) induces NETosis by releasing neutrophil extracellular traps (NETs), while nanometer-sized GO (nGO) elicits neutrophil degranulation. The neutrophils’ defensive behavior is accompanied with generation of reactive oxygen species and activation of p-ERK and p-Akt kinases. We unveil that mGO-induced NETosis is NADPH oxidase (NOX)-independent, while nGO-triggered degranulation is NOX-dependent. Furthermore, myeloperoxidase (MPO) is identified to be a determinant mediator despite distinct neutrophil phenotypes in the biodegradation. Neutrophils release NETs comprising of MPO upon activated with mGO, while MPO is secreted via nGO induced-degranulation. Moreover, the binding energy between MPO and GO is calculated to be 69.8728 kJ·mol− 1, which indicates that electrostatic interactions mainly cause the spontaneous binding process in a spatial distance of 9.2 Å. Meanwhile, the central enzymatic biodegradation is found to occur at oxygenic active sites and defects on GO. Mass spectrometry analysis deciphers the degradation products are biocompatible molecules like flavonoids and polyphenols. Our study provides fundamental evidence and practical guidance for functional biomaterial development in sustainable nanotechnology, including but not limited to vaccine adjuvant and drug carrier.

The abuse application of glyphosate can result in a potential hazard for environment and human, however its ultrasensitive detection remains challenging. Herein, a Cu

Central nervous system diseases commonly occur with the destruction of the blood-brain barrier. As a primary cause of morbidity and mortality, stroke remains unpredictable and lacks cellular biomarkers that accurately quantify its occurrence and development. Here, we identify NeuN + /CD45 − /DAPI + phenotype nonblood cells in the peripheral blood of mice subjected to middle cerebral artery occlusion (MCAO) and stroke patients. Since NeuN is a specific marker of neural cells, we term these newly identified cells as circulating neural cells (CNCs). We find that the enumeration of CNCs in the blood is significantly associated with the severity of brain damage in MCAO mice ( p < 0.05 ). Meanwhile, the number of CNCs is significantly higher in stroke patients than in negative subjects ( p < 0.0001 ). These findings suggest that the amount of CNCs in circulation may serve as a clinical indicator for the real-time prognosis and progression monitor of the occurrence and development of ischemic stroke and other nervous system disease.

The abuse application of glyphosate can result in a potential hazard for environment and human, however its ultrasensitive detection remains challenging. Herein, a Cu2+ modulated DNA-templated silver nanoclusters (DNA-AgNCs) sensor was constructed to sensitively determine glyphosate based on the turn-on fluorescence strategy. The fluorescence quenching of DNA-AgNCs occurred with the existence of Cu2+. Upon the presence of glyphosate, the functional groups on the surface of glyphosate could chelate with Cu2+, following the fluorescence recovery of DNA-AgNCs. Through the stoichiometric methods, we unveil that Cu2+-trigged fluorescence quenching mode is a combination of static and dynamic quenching with the static mode being predominant. In DNA-AgNCs/Cu2+ system, the carboxylate, amine, and phosphonate groups of glyphosate interact with Cu2+ through chelation, in which the carboxylate oxygen, the phosphonate oxygen atoms, and the monoprotonated secondary amine nitrogen atom and Cu2+ form chelate rings. This fluorescence sensor showed a desired linearity of glyphosate analysis under the optimum conditions, ranging from 15 to 100 μg/L with a low detection down to 5 μg/L. Moreover, the proposed sensor was successfully utilized to measure glyphosate in real samples, indicating a promising application in pesticide residues detection.

Nanodiamonds (NDs) have been extensively explored in biomedical applications due to their favorable properties, such as biocompatibilities and diverse surface electrostatics that allow rapid conjugations with drugs. Enhancing chemotherapeutic efficacy through optimized nanodiamond drug delivery system would further improve the treatment of cancers. In this study, carboxylated nanodiamonds and original nanodiamonds were involved to explore the maximum efficiency in binding and release capability of doxorubicin (DOX). The binding ratio of NDs and DOX, pH values, and release time were optimized. Notably, an NDX synthesized in 2 mL ND, 5 mL DOX, and 0.061 mL NaOH exhibits a ~63.4% (31.7 wt%) binding efficiency, and NDX release profile quantified in acidic environment exhibits ~99.9% release efficiency of DOX. The present work establishes a foundation for future applications in drug delivery system for enhancing chemotherapy efficacy.

DNA hydrogel garnered increasing attention in the sensing and medical field owing to its native biocompatibility and mechanical stability. While electrochemistry serves as a quantitative and sensitive detection technique, electrochemical DNA hydrogel biosensor is rarely reported. Here, for the first time, we report an electrochemical biosensor based on hybrid DNA hydrogel immobilized on indium tin oxide/polyethylene terephthalate (ITO/PET) electrode for the detection of lung cancer-specific microRNA, miR-21. The biosensor is capable of detecting miR-21 at a concentration as low as 5 nM (1 pmol) and linear read-out from 10 nM to 50 μM. Ferrocene-tagged recognition probes were cross-linked with DNAs grafted on the polyacrylamide backbones to form the hybrid DNA hydrogel, which was further immobilized on 3-(trimethoxysilyl)propyl methacrylate (KH 570) treated ITO electrode. When the recognition probe was hybridized with the target miR-21, the hydrogel dissolved, producing a loss of ferrocene tags and a reduction in current, detected by Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV). The material characteristics of the biosensor were verified using contact angle meter and Energy Dispersive Spectrometer (EDS). This novel biosensor holds great promise in early sensitive clinical diagnosis for a variety of cancer-specific biomarkers due to the flexible sequence design of the recognition probe.

The boosting exploitation of graphene oxide (GO) increases exposure risk to human beings. However, as primary defender in the first immune line, neutrophils' mechanism of defensive behavior towards GO remains unclear. Herein, we discover that neutrophils recognize and defensively degrade GO in a lateral dimension dependent manner. The micrometer-sized GO (mGO) induces NETosis by releasing neutrophil extracellular traps (NETs), while nanometer-sized GO (nGO) elicits neutrophil degranulation. The two neutrophils' defensive behaviors are accompanied with generation of reactive oxygen species and activation of p-ERK and p-Akt kinases. We unveil that mGO-induced NETosis is NADPH oxidase (NOX)-independent while nGO-triggered degranulation is NOX-dependent. Furthermore, myeloperoxidase (MPO) is identified to be a determinant mediator despite distinct neutrophil phenotypes in the biodegradation. Neutrophils release NETs comprising of MPO upon activated with mGO, while MPO is secreted via nGO induced-degranulation. Moreover, the binding energy between MPO and GO is calculated to be 69.8728 kJ·mol-1 , which indicates that electrostatic interactions mainly cause the spontaneous binding process in a spatial distance of 9.2 A. Meanwhile, the central enzymatic biodegradation is found to occur at oxygenic active sites and defects on GO. Mass spectrometry analysis deciphers the degradation products are biocompatible molecules like flavonoids and polyphenols. Our study provides fundamental evidence and practical guidance for nanotechnology based on GO, including but not limited to vaccine adjuvant, implantable devices and energy storage. This article is protected by copyright. All rights reserved.

Flexible sensors based on multi-walled carbon nanotubes (MWCNTs) and their composite materials are widely applied to develop strain sensors that generate a signal as a function of the physical or mechanical deformation. However, on-site analysis of the electrochemical active substances with the flexible electrodes during the biological or medical processes remains a great challenge for current electrochemical technologies. Herein, a universal three-electrode module, adaptable of the synchronous electrochemical analysis in practical applications has been developed with the MWCNTs and polydimethylsiloxane (PDMS) flexible film as the working and the counter electrodes and a painted silver-paste line as the pseudo-reference electrode. The MWCNT/PDMS flexible eletrodes capable of being customized into various sizes, shapes and geometries according to the detection environmental requirements, were prepared through a simple molding transfer method and endowed the on-site electrochemical analysis module with a great potential on the curved surface or in the repeatedly bending situation. Finally, in a 24-well plate and a medical drip which were equipped with the developed three-electrode module, the concentrations of dopamine (DA), uric acid (UA) and nicotinamide adenine dinucleotide (NADH) were successfully detected by Differential Pulse Voltammetry (DPV) with robust stability and repeatability. The technique developed in this study offers an accurate and cost-effective on-site electrochemical analysis system that can be modularly adjoined into various biological and medical applications.

Recent reports have indicated that aberrant expression of microRNAs is highly correlated with occurrence of lung cancer. Therefore, highly sensitive detection of lung cancer specific microRNAs provides an attractive approach in lung cancer early diagnostics. Herein, we designed 3D DNA origami structure that enables electrochemical detection of lung cancer related microRNAs. The 3D DNA origami structure is constituted of a ferrocene-tagged DNA of stem-loop structure combined with a thiolated tetrahedron DNA nanostructure at the bottom. The top portion hybridized with the lung cancer correlated microRNA, while the bottom portion was self-assembled on gold disk electrode surface, which was modified with gold nanoparticles (Au NPs) and blocked with mercaptoethanol (MCH). The preparation process and the performance of the proposed electrochemical genosensor were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Under the optimal conditions, the developed genosensor had a detection limit of 10 pM and a good linearity with microRNA concentration ranging from 100 pM to 1 µM, which showed a great potential in highly sensitive clinical cancer diagnosis application.

Lead ion is one of the most hazardous and ubiquitous heavy metal pollutants and poses an increasing threat to the environment and human health. This necessitates rapid and selective detection and/or removal of lead ions from various soil and water resources. Recently, we identified several Pb 2+ binding peptides via phage display technique coupled with chromatographic biopanning ( Nian et al., 2010 ) where a heptapeptide (TNTLSNN) capable of recognizing Pb 2+ with high affinity and specificity evolved. In the present study, an electrochemical sensor harnessing this Pb 2+ affinity peptide as a probe on a porous gold electrode was developed. The three dimensional porous gold electrode was obtained from electrochemical deposition using the dynamic hydrogen bubble template method. A thin layer of poly(thiophene acetic acid) (PTAA) was coated on the porous gold surface. The Pb 2+ recognizing peptide was immobilized via amide linkage on the PTAA. The developed biosensor was demonstrated to be fast, selective and reproducible in Pb 2+ detection, exhibiting Pb 2+ -specific peak current values around −0.15 V in a broad concentration range (1−1×10 7 nM) in 10 min despite the repeated use after regeneration.

Neatly arranged gold nanoparticles (AuNPs) were directly electrodeposited on an electrochemically polymerized self-assembled monolayer (SAM) of thiol-functionalized 3,4-ethylenedioxythiophene (EDOT) derivative, EDTMSHA. A thiolated single-stranded DNA (ssDNA) aptamer with high specificity to LPS was immobilized on the AuNPs/conducting polymer composite film, serving as sensing platform for LPS detection. Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), scanning electron microscope (SEM), and atomic force microscopy (AFM) were utilized to characterize the modification and detection processes. The electron transfer resistance was found to have a linear relationship with LPS concentration from 0.1 pg/mL to 1 ng/mL.

Lipopolysaccharide (LPS), also known as endotoxin, triggers a fatal septic shock; therefore, fast and accurate detection of LPS from a complex milieu is of primary importance. Several LPS affinity binders have been reported so far but few of them have proved their efficacy in developing electrochemical sensors capable of selectively detecting LPS from crude biological liquors. In this study, we identified 10 different single-stranded DNA aptamers showing specific affinity to LPS with dissociation constants ( K d ) in the nanomolar range using a NECEEM-based non-SELEX method. Based on the sequence and secondary structure analysis of the LPS binding aptamers, an aptamer exhibiting the highest affinity to LPS (i.e., B2) was selected to construct an impedance biosensor on a gold surface. The developed electrochemical aptasensor showed excellent sensitivity and specificity in the linear detection range from 0.01 to 1 ng/mL of LPS with significantly reduced detection time compared with the traditional Limulus amoebocyte lysate (LAL) assay.

An alkylthiol functionalized monomer based on 3,4-ethylenedioxythiophene methanol (EDTM), namely 3-((2,3-dihydrothieno[3,4-b][1,4]dioxin-2-yl)methoxy)propane-1-thiol (EDTMSHA), was synthesized and confirmed by 1H NMR, 13C NMR, and gas chromatography mass spectroscopy (GC/MS). The new monomer successfully produced a self-assembled monolayer (SAM), which was deposited on a gold disc electrode and then electrochemically polymerized. The SAM formation of the new conducting polymer films exhibited relatively low oxidation potentials with a well defined reversible and stable electrochemical behavior compared to conventional electropolymerized film.

Dihydronicotinamide adenine dinucleotide (NADH) is a major biomarker correlated with lethal diseases such as cancers and bacterial infection. Herein, we report a graphene-DNA tetrahedron-gold nanoparticle modified gold disk electrode for highly sensitive NADH detection. By assembling the DNA tetrahedron/graphene composite film on the gold disk electrode surface which prior harnessed electrochemical deposition of gold nanoparticles to enhance the effective surface area, the oxidation potential of NADH was substantially decreased to 0.28 V (vs. Ag/AgCl) and surface fouling effects were successfully eliminated. Furthermore, the lower detection limit of NADH by the presented platform was reduced down to 1 fM, with an upper limit of 10 pM. Both the regeneration and selectivity of composite film-modified electrode are investigated and proved to be robust. The novel sensor developed here could serve as a highly sensitive probe for NADH detection, which would further benefit the field of NADH related disease diagnostics.

Endotoxin, present in the outer membrane of all gram-negative bacteria, can pose serious risks to human health, from irreversible shock to death. Therefore, it is essential to develop sensitive, accurate, and rapid methods for its detection. The rabbit pyrogen test is the first standard technique for endotoxin detection and, nowadays, has been replaced by the Limulus Amoebocyte Lysate test, which is the most popular detection technique for endotoxin. With in-depth understanding of endotoxin, biosensors based on endotoxin-sensing components are promising alternatives to pursue in developing low-cost, easy-operation, and fast-response endotoxin detection techniques. This article summarizes the recent advances of endotoxin detection methods with a particular emphasis on optical and electrochemical biosensors based on various sensing elements ranging from nature biomolecules to artificial materials. As the research and technological revolution continues, the highly integrated and miniaturized commercial devices for sensitively and reliably detecting endotoxin will provide a wide range of applications in people's daily life.

Using a single-stranded DNA (ssDNA) aptamer exhibiting high binding affinity (Kd = 12 nM) to endotoxin as a probe, an impedance sensor where aptamer-conjugated gold nanoparticles (AuNPs) were electrochemically deposited on a gold electrode was fabricated and its performance in regard to endotoxin detection assessed. AuNPs have been employed widely as biosensors because of their unique physical and chemical properties. In order to maximize the performance of the impedance aptasensor on endotoxin detection, some critical factors affecting aptamer conjugation to AuNPs and target recognition ability (i.e. concentrations of aptamer coupled with AuNPs, pH, ion strength and cation effect at the time of aptamer–endotoxin interaction) were optimized. Electrochemical impendence spectroscopy, cyclic voltametry, atomic force microscope, scanning electron microscope and quartz crystal microbalance were employed to characterize all the modification/detection procedures during the sensor fabrication. The developed aptasensor showed a broad linear dynamic detection range (0.01–10.24 ng/ml) with a very low detection limit for endotoxin (0.005 ng/ml), despite the presence of several biomolecules (e.g. plasmid DNA, RNA, serum albumin, Glc and sucrose) known to interfere with other endotoxin assays. The demonstrated aptasensor required a detection time of only 10 min, providing a simple and fast analytical method to specifically detect endotoxin from complex biological liqors.

Lipopolysaccharide (LPS) often referred to endotoxin is an undesirable impurity frequently entrained with various recombinant protein therapeutics and plasmid DNA (pDNA) vaccines of bacterial origin. The inherent toxicities (e.g. fever, hypotension, shock and death) of LPS render its early and sensitive detection essential for several biological assays and/or parenteral administrations of biotherapeutics. In this study, an electrochemical biosensor using an LPS specific single stranded DNA (ssDNA) aptamer as a probe was developed. Amine-terminated aptamer exhibiting high affinity (K(d)=11.9 nM) to LPS was immobilized on a gold electrode using 3-mercaptopropionic acid (MPA) as a linker. Each step of the modification process was characterized by cyclic voltammetry (CV) and electrochemical impendence spectroscopy (EIS). A good linear relationship of the changes in the charge-transfer resistance (ΔR(et)) and the logarithmic value of LPS concentration was demonstrated in a broad dynamic detection range of 0.001-1 ng/ml. Furthermore, the aptasensor showed a high selectivity to LPS despite the presence of pDNA, RNA and bovine serum albumin (BSA) and could be regenerated in low pH condition, offering a promising option for detecting LPS often present in a complex milieu.