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.
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.
In this work, we demonstrated a silicon nanowire (SiNW) biosensing platform capable of simultaneously identifying different Dengue serotypes on a single sensing chip. Four peptide nucleic acids (PNAs), specific to each Dengue serotypes (DENV-1 to DENV-4), were spotted on different areas of the SiNW array surface, and the covalently immobilized PNA probes were then interacted with different Dengue serotypes target to establish the specificity of detection. Detection scheme is based on the changes in resistances due to accumulation of negative charges contributed by the hybridized DNA target. The results show that resistance changes only occur in regions where the Dengue target hybridizes with its complementary probe. What is more, a mixture of two different Dengue serotypes obtained from a one-step duplex RT-PCR was applied to the multiplex SiNW surface to validate SiNW capability to identify multiple Dengue serotypes on a single sensing platform. Through this study, we have established the multiplex SiNW biosensor as a promising device to detect multiple Dengue infections with high specificity.
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