Microvesicle detection by a reduced graphene oxide field-effect transistor biosensor based on a membrane biotinylation strategy.

Unlike other extracellular vesicle (EV) subtypes such as exosomes, the lack of well-defined universal markers on the surface of microvesicles (MVs) has led to difficulty in the detection of the entire MV population. To design a universal MV detection method, we reported highly sensitive electrical detection of MVs using a reduced graphene oxide (RGO)-based field-effect transistor (FET) biosensor by the introduction of a membrane biotinylation strategy in this work. Biotinylated MVs (B-MVs) were obtained by supplying the culture medium with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG-biotin) while cultivating the cells. Excellent biotinylation efficiency of MVs (92.6%) was then realized. A streptavidin (SA) probe was subsequently modified onto the channel surface of the as-fabricated RGO-based FET device, which was capable of specifically recognizing B-MVs due to the high affinity between SA and biotin in a 1 : 4 recognition format. The results showed that the RGO-based FET biosensor could detect B-MVs in a wide range from 105 particles per mL to 109 particles per mL with a low detection limit down to 20 particles per µL, which was the lowest value compared with other previously reported results. This platform also allowed distinguishing B-MVs from other unbiotinylated EV types such as MVs and exosomes, exhibiting excellent specificity. Moreover, this FET biosensor demonstrated the capability of detecting B-MVs derived from different cell lines including cancer cells and normal cells, indicating its versatility and potential applications in the biomedical field.

Gold nanoparticles-decorated graphene field-effect transistor biosensor for femtomolar MicroRNA detection.

Early detection is proven to be the best chance for successful cancer treatment. MiRNAs, as ideal biomarkers, can identify cancer in the early stage. Therefore, development of highly sensitive and selective detection methods for miRNA is still anticipated. Here we report on a gold nanoparticles (AuNPs)-decorated graphene field-effect transistor (FET) biosensor for highly sensitive, selective and label-free detection of miRNA. The AuNPs-decorated graphene FET biosensor was fabricated by drop-casting the reduced graphene oxide (R-GO) suspension onto the sensor surface, and subsequently decorating AuNPs onto the surface of R-GO. After peptide nucleic acid (PNA) probe was immobilized on the AuNPs surface, miRNA detection was carried out via PNA-miRNA hybridization. It was found that the developed FET biosensor was able to achieve a detection limit as low as 10 fM. In addition, the biosensor enabled an accurate distinction of complementary miRNA from one-base mismatched miRNA and noncomplementary miRNA. What's more, this highly sensitive and selective assay was also applied to the detection of miRNA in serum samples, making it a potential method for diagnosis of gene-related diseases.

Silicon nanowire biosensor for highly sensitive and multiplexed detection of oral squamous cell carcinoma biomarkers in saliva.

Silicon nanowire (SiNW) field-effect transistor (FET) biosensors have already been used as powerful sensors for the direct detection of disease-related biomarkers. However, the multiplexed detection of biomarkers in real samples is still challenging. Interleukin 8 (IL-8) and tumor necrosis factor α (TNF-α) are two typical biomarkers of oral squamous cell carcinoma (OSCC). In this study, we developed a multiplexed detection methodology for IL-8 and TNF-α detection in saliva using SiNW FET biosensors. We fabricated the SiNW FET sensors using a top-down lithography fabrication technique. Subsequently, we achieved the multiplexed detection of two biomarkers in saliva by specific recognition of the two biomarkers with their corresponding antibodies, which were modified on the SiNW. The established method was found to have a limit of detection as low as 10 fg/mL in 1 × PBS as well as 100 fg/mL in artificial saliva. Because of its advantages, including label-free and multiplexed detection, non-invasive analysis, highly sensitive and specific determination, the proposed method is expected to be widely used for the early diagnosis of OSCC.

Bioluminescence imaging of hollow fibers in living animals: its application in monitoring molecular pathways.

We have applied noninvasive optical imaging technology to the in vivo hollow fiber assay, using tumor cell lines in which optical reporters are expressed in response to activation/inhibition of a specific molecular pathway. In vivo noninvasive imaging of molecular pathways in cells within hollow fibers enables a rapid and accurate evaluation of drug targets and provides useful insights to guide novel drug discovery. In this protocol we show, as an example, that a luciferase reporter, driven by the responsive element of nuclear factor NF-kappaB, was induced in cells within hollow fibers implanted in living mice, and a detailed procedure for in vivo bioluminescence imaging of hollow fibers is described. This approach can, in principle, be applied to image any molecular pathways of interest when appropriate reporter cells are generated. Hollow fiber encapsulation and implantation takes 2 d, and in vivo validation of reporter takes 1-2 weeks.