Integrated FET sensing microsystem for specific detection of pancreatic cancer exosomal miRNA10b.

Tumor-derived exosome (TD-Ex) serves as a crucial early diagnostic biomarker of pancreatic cancer (PC). However, accurate identification of TD-Ex from PC is still a challenging work. In this paper, a detection microsystem that integrates magnetic separation and FET biosensor is developed, which is capable of selectively separating TD-Ex of PC from the plasma and detecting exosomal miRNA10b in a sensitive and specific manner. The magnetic beads were functionalized with dual antibody (GPC-1 antibody and EpCAM antibody), enabling selective recognition and capture of PC-derived exosomes. On the other hand, a peptide nucleic acid (PNA)- functionalized reduced graphene oxide field-effect transistor (RGO FET) biosensor was subsequently utilized to detect the exosomal miRNA10b, which is highly expressed in PC- derived exosomes. This system could achieve a low detection limit down to 78 fM, and selectively identify miRNA10b from single-base mismatched miRNA. In addition, 40 clinical plasma samples were tested with this microsystem, and the results indicate that it could effectively distinguish PC patients from healthy individuals. The assay combines specific capture and enrichment of PC-derived exosomes with sensitive and selective detection of exosomal miRNA, showing its potential to be used as an effective scheme for PC early diagnosis.

Peptide Nucleic Acid-Functionalized Nanochannel Biosensor for the Highly Sensitive Detection of Tumor Exosomal MicroRNA.

Compared with free miRNAs in blood, miRNAs in exosomes have higher abundance and stability. Therefore, miRNAs in exosomes can be regarded as an ideal tumor marker for early cancer diagnosis. Here, a peptide nucleic acid (PNA)-functionalized nanochannel biosensor for the ultrasensitive and specific detection of tumor exosomal miRNAs is proposed. After PNA was covalently bound to the inner surface of the nanochannels, the detection of tumor exosomal miRNAs was achieved by the charge changes on the surface of nanochannels before and after hybridization (PNA-miRNA). Due to the neutral characteristics of PNA, the efficiency of PNA-miRNA hybridization was improved by significantly reducing the background signal. This biosensor could not only specifically distinguish target miRNA-10b from single-base mismatched miRNA but also achieve a detection limit as low as 75 aM. Moreover, the biosensor was further used to detect exosomal miRNA-10b derived from pancreatic cancer cells and normal pancreatic cells. The results indicate that this biosensor could effectively distinguish pancreatic cancer tumor-derived exosomes from the normal control group, and the detection results show good consistency with those of the quantitative reverse-transcription polymerase chain reaction method. In addition, the biosensor was used to detect exosomal miRNA-10b in clinical plasma samples, and it was found that the content of exosomal miRNA-10b in cancer patients was generally higher than that of healthy individuals, proving that the method is expected to be applied for the early diagnosis of cancer.

Synergy of Peptide-Nucleic Acid and Spherical Nucleic Acid Enabled Quantitative and Specific Detection of Tumor Exosomal MicroRNA.

Exosomal microRNAs are essential in intercellular communications and disease progression, yet it remains challenging to quantify the expression level due to their small size and low abundance in blood. Here, we report a "sandwich" electrochemical exosomal microRNA sensor (SEEmiR) to detect target microRNA with high sensitivity and specificity. In SEEmiR, neutrally charged peptide nucleic acid (PNA) enables kinetically favorable hybridization with the microRNA target relative to negatively charged DNA, particularly in a short sequence (10 nt). More importantly, this property allows PNA to cooperate with a spherical nucleic acid (SNA) nanoprobe that heavily loads with oligonucleotide-adsorbed electroactive tags to enhance detection sensitivity and specificity. Such a PNA-microRNA-SNA sandwich construct is able to minimize the background noise via PNA, thereby maximizing the SNA-mediated signal amplification in electrostatic adsorption-based SEEmiR. The synergy between PNA and SNA makes the SEEmiR sensor able to achieve a broad dynamic range (from 100 aM to 1 nM) with a detection limit down to 49 aM (2 orders of magnitude lower than that without SNA) and capable of distinguishing a single-base mismatch. This ultrasensitive sensor provides label-free and enzyme-independent microRNA detection in cell lysates, unpurified tumor exosomal lysates, cancer patients' blood, and accurately differentiates the patients with breast cancer from the healthy ones, suggesting its potential as a promising tool in cancer diagnostics.