Lab in a Tube: Ultrasensitive Detection of MicroRNAs at the Single-Cell Level and in Breast Cancer Patients Using Quadratic Isothermal Amplification

Abstract

Through rational design of a functional molecular probe with high sequence specificity that takes advantage of sensitive isothermal amplification with simple operation, we developed a one-pot hairpin-mediated quadratic enzymatic amplification strategy for microRNA (miRNA) detection. Our method exhibits ultrahigh sensitivity toward miR-21 with detection limits of 10 fM at 37 °C and 1 aM at 4 °C, which corresponds to nine strands of miR-21 in a 15 μL sample, and it is capable of distinguishing among miRNA family members. More importantly, the proposed approach is also sensitive and selective when applied to crude extractions from MCF-7 and PC3 cell lines and even patient tissues from intraductal carcinoma and invasive ductal carcinoma of the breast.

Polymerase/nicking enzyme synergetic isothermal quadratic DNA machine and its application for one-step amplified biosensing of lead (II) ions at femtomole level and DNA methyltransferase

“…This assay time and the high signal-to-noise ratio (18-fold) are superior to those of the previously reported methods based on cascade amplification. [21][22][23][24][25] …”

“…This assay time and the high signal-to-noise ratio (18-fold) are superior to those of the previously reported methods based on cascade amplification. [21][22][23][24][25] Analytical performance for Pb 2+ detection based on ESQM For the detection of Pb 2+ , a DNAzyme (GR-5 DNAzyme) is involved in the proposed biosensor as a Pb 2+ recognition unit. GR-5 DNAzyme is a functionalized nucleic acid with catalytic activity, cleaving the Figure 2 The fluorescence responses of the proposed sensing system (a) and basic SDA system (b) in the presence of 10 nM SDA primer, with corresponding backgrounds without SDA primer (c, d), respectively.…”

Self-assembled silk fibroin nanoparticles loaded with binary drugs in the treatment of breast carcinoma

“…[1][2][3] Many strategies have been developed for cancer diagnosis 4 and therapy, such as immunotherapy, thermal therapy, phototherapy, surgery, gene therapy, chemotherapy, and radiotherapy. [5][6][7][8][9][10][11] Each of these methods has its own advantages and disadvantages.…”

Chip-based visual detection of microRNA using DNA-functionalized gold nanoparticles

“…Detecting miRNAs is important in the early diagnosis of diseases as well as finding new drug targets. However, due to their short length, low abundance, and high sequence similarity, studies on miRNAs is challenging (Duan et al, 2013;Koshiol et al, 2010;Leshkowitz et al, 2013;Yang et al, 2009). Northern blotting is the most commonly used method to detect miRNAs (Cissell and Deo, 2009;Streit et al, 2008); however, it has the disadvantages of low sensitivity, complex, and time consuming, thereby limiting its utility in a clinical setting.…”

“…Northern blotting is the most commonly used method to detect miRNAs (Cissell and Deo, 2009;Streit et al, 2008); however, it has the disadvantages of low sensitivity, complex, and time consuming, thereby limiting its utility in a clinical setting. Real-time PCR can cover a broad dynamic range with high sensitivity, but it requires precise temperature control and the short length of miRNAs makes the primer design very difficult (Duan et al, 2013;Git et al, 2010;Kroh et al, 2010;Ren et al, 2013). Electrochemical based methods have high sensitivity (Gao and Peng, 2011;Gao and Yu, 2007;Wen et al, 2012;Yin et al, 2012).…”