Dual-Aptamer Modified Graphene Field-Effect Transistor Nanosensor for Label-Free and Specific Detection of Hepatocellular Carcinoma-Derived Microvesicles

Abstract

Cancerous microvesicles (MVs), which are heterogeneous membrane-bound nanovesicles shed from the surfaces of cancer cells into the extracellular environment, have been widely recognized as promising “biofingerprints” for various cancers. High-performance identification of cancerous MVs plays a vital role in the early diagnosis of cancer, yet it is still technically challenging. Herein, we report a gold nanoparticle (AuNP)-decorated, dual-aptamer modified reduced graphene oxide (RGO) field-effect transistor (AAP-GFET) nanosensor for the label-free, specific, and sensitive quantification of HepG2 cell-derived MVs (HepG2-MVs). After GFET chips were fabricated, AuNPs were then decorated on the RGO surface. For specific capture and detection of HepG2-MVs, both sulfhydrylated HepG2 cell specific TLS11a aptamer (AptTLS11a) and epithelial cell adhesion molecule aptamer (AptEpCAM) were immobilized on the AuNP surface through an Au–S bond. This developed nanosensor delivered a broad linear dynamic range from 6 × 105 to 6 × 109 particles/mL and achieved a high sensitivity of 84 particles/μL for HepG2-MVs detection. Moreover, this AAP-GFET platform was able to distinguish HepG2-MVs from other liver cancer-related serum proteins (such as AFP and CEA) and MVs derived from human normal cells and other cancer cells of lung, pancreas, and prostate, suggesting its excellent method specificity. Compared with those modified with a single type of aptamer alone (AptTLS11a or AptEpCAM), such an AAP-GFET nanosensor showed greatly enhanced signals, suggesting that the dual-aptamer-based bio–nano interface was uniquely designed and could realize more sensitive quantification of HepG2-MVs. Using this platform to detect HepG2-MVs in clinical blood samples, we found that there were significant differences between healthy controls and hepatocellular carcinoma (HCC) patients, indicating its great potential in early HCC diagnosis.

Highly Sensitive Uric Acid Detection Based on a Graphene Chemoresistor and Magnetic Beads

“…Graphene biosensors exhibit the advantages of miniaturization, a low limit of detection, high sensitivity, and simplicity of design. Several research groups have reported the use of graphene sensors for the measurement and detection of chemical and biological components and target biomarkers for diseases, including cancer markers, DNA, and glucose [ 16 , 17 ]. However, the above-mentioned applications directly immobilize and functionalize receptor molecules on the graphene surface.…”

Graphene field-effect transistors as bioanalytical sensors: design, operation and performance

“…Additional strategies in the design of the interface have also been reported to enhance signal strength, such as the addition of protective layers 162 or addition of gold nanoparticles. 120,198 Importantly, the detailed structure of the interface is often weakly characterized: in particular, the density of probe molecules on graphene is usually poorly controlled and often unknown, in large part because it is difficult to measure experimentally. In consequence, there is little quantitative knowledge on the correlation between interface design and the resulting LOD (or any other performance metrics).…”

“…In such assays, target molecules are most commonly diluted in blank saline buffer, 42,88,90,117 sometimes with a calibrated mix of interfering species. 53,55,83,91,118 These calibration assays are usually presented as a proof of concept for the sensors, and they are sometimes used as positive controls before assays on cell culture samples, 51,56 clinical samples 55,115,121,136,198 or other environmental samples, 46,123 in which the concentration is either unknown or measured with another detection technique to compare results. For instance, Wang et al 121 were the first group to measure the concentration of lead ions in real blood samples with GFET sensors, using a calibration with positive controls in buffer.…”

“…the possibility to make successive analyses on the same device, as well as shelf-life of the sensors are other important parameters in technology maturation, and have been tested in some recent studies. For example, reusability has been tested by Wu et al 198 by performing successive binding-unbinding cycles with the target and measuring the evolution in the strength of signal, finding a conservation of signal of 94% when comparing the first to the last cycle. Some groups also made assessments of shelf-life by measuring the drift of current over time for multiple concentrations of target 119 or by repeating experiments after storage time.…”

Immobilization of Target-Bound Aptamer on Field Effect Transistor Biosensor to Improve Sensitivity for Detection of Uncharged Cortisol

“…To overcome this challenge, aptamers have been applied as the receptors of FET biosensors, utilizing their ability to recognize small molecules and conformational changeability. [5][6][7] Aptamers, which are only several nanometers in size, can be artificially designed and selected from a large oligonucleotide library via the systematic evolution of ligands by exponential enrichment (SELEX). 8,9 In particular, the ease of the structural design of aptamers allows for the introduction of conformational changeability with the binding of the target molecule.…”