Allosteric aptasensor-initiated target cycling and transcription amplification of light-up RNA aptamer for sensitive detection of protein

Abstract

The early detection of biomarker proteins in clinical samples is of great significance for the diagnosis of diseases. However, it is still a challenge to detect low-concentration protein. Herein, a label-free aptamer-based amplification assay, termed the ATC-TA system, that allows fluorescence detection of very low numbers of protein without time-consuming washing steps and pre-treatment was developed. The target induces a conformational change in the allosteric aptasensor, triggers the target cycling and transcription amplification, and ultimately converts the input of the target protein into the output of the light-up aptamer (R-Pepper). It exhibits ultrahigh sensitivity with a detection limit of 5.62 fM at 37 ℃ and the accuracy is comparable to conventional ELISA. ATC-TA has potential application for the detection of endogenous PDGF-BB in serum samples to distinguish tumor mice from healthy mice at an early stage. It also successfully detects exogenous SARS-CoV-2 spike proteins in human serum. Therefore, this high-sensitive, universality, easy-to-operate and cost-effective biosensing platform holds great clinical application potential in early clinical diagnosis.

Keywords: Allosteric aptasensor; Light-up RNA aptamer; Protein detection; Target cycling signal amplification; Transcription amplification.

Fig. 1

Schematic illustration of the allosteric aptasensor-initiated target cycling and transcription amplification of RNA aptamer for protein detection.

Fig. 2

The feasibility analysis of the ATC-TA system. A. Schematic illustrates the ELISA principle of PDGF-BB binding to the biotin-labeled Aa. B. ELISA reveals the ability of Aa to bind human PDGF-BB. Apt: aptamer of PDGF-BB. RS: random sequence. Blank: without DNA. All the P values were determined using paired t-test. * * P ≤ 0.01. C. Fluorescence intensities of Broccoli (1 μM) binding to DFHBI (10 µM) and Pepper (1 μM) binding to HBC (10 µM). The excitation and emission wavelengths are 460 nm and 500 nm for DFHBI. D. Comparison of the structure and sequence of Pepper, R-Pepper, Pepper-11, R-Pepper-11. The sequence binding to HBC is labelled in orange. R-Pepper and R-Pepper-11 are in the black box. E. The fluorescence peak density of those light-up aptamers in the presence of HBC, respectively. F. Aa11 and Tp8 do not extend without PDGF-BB. G. Bst DNA polymerase extends on the Aa11-Tp8 duplex to yield double-stranded DNA with PDGF-BB. H. T7 RNA polymerase recognizes the T7 promoter sequence and transcribes R-pepper-11. I. 16% native PAGE analysis of experimental feasibility. Lane 1: DNA ladder; Lane 2: Aa11; Lane 3: Tp8; Lane 4: Aa11 + Tp8 + Bst; Lane 5: Aa11 + PDGF-BB+ Tp8 + Bst; Lane 6: Aa11 + Tp8 + Bst+ T7; Lane 7: PDGF-BB+ Aa11 + Tp8 + Bst+ T7; The synthetic dsDNA (120 bp, Lane 8) and the transcribed R-Pepper-11 (Lane 9) were also used as the marker. J. Fluorescence spectra of each step of the ATC-TA system. Error bars were shown as means ± S.D., n = 3, triplicate.

Fig. 3

Optimization of reaction parameters. A. The structure and sequence of pre-Aa10 and Aa10 were predicted using UNAFold. Carmine: aptamer; Orange: T7 promoter; Blue: extender. Dark green: GT bases. B. Optimizing the stem length of Aa. C. Comparison of different Tp with varied lengths which are complementary with Aa. D. The concentration of Aa10 tested in ATC-TA. Error bars were shown as means ± S.D., n = 3, triplicate.

Fig. 4

Performance of ATC-TA system. A. Fluorescence spectra of ATC-TA in response to different concentrations of PDGF-BB. Blank: without PDGF-BB. B. Calibrated curve of the average fluorescence intensity at 514 nm. Inset shows the linear responses at low PDGF-BB concentrations. C. Selectivity evaluation of ATC-TA system. Mixed: the mix of PDGF-BB, PDGF-AB, PDGF-AA, TNF-α IgG and BSA, each at a concentration of 5 nM. D. Colorimetric detection of PDGF-BB with different concentrations. E. Real-time detection of PDGF-BB at different concentrations by ATC-TA. F. Linear analysis of real-time detection of PDGF-BB at different times. Error bars were shown as means ± S.D., n = 3, triplicate.

Fig. 5

Detection of the PDGF-BB in serum. A. Fluorescence peak density of PDGF-BB in different dilutions of human serum solution. B. ELISA reveals the ability of Aa to bind mouse PDGF-BB. The P values were determined using paired t-test. * P ≤ 0.05. C. Schematic illustration for the construction of mouse MC38 colon tumor models and blood collection at the indicated time. 59 mm³ and 97 mm³ represent the average tumor volume at the indicated time, respectively. D. Mouse PDGF-BB expression curves versus time of six infected mice (n = 6) and six healthy mice ((n = 6, dash-dot lines), with the average trace (red line, black line) shown. E. Histogram shows the difference in mouse PDGF-BB content between model mice and the healthy group at the indicated time. Day 5 for the healthy Group. All the P values were determined using paired t-test. * ** P ≤ 0.001; * ** * P ≤ 0.0001. All experiments were repeated three times. Error bars were shown as means ± S.D., n = 3, triplicate.

Fig. 6

Detection of the SARS-CoV-2 spike protein in human serum. A. Fluorescence spectra of ATC-TA in response to different concentrations of the SARS-CoV-2 spike protein. Blank: without spike protein. B. The calibrated curve was made according to the fluorescence spectra. C. Selectivity study of ATC-TA system against the spike protein of coronaviruses. D. Fluorescence peak intensity to assess the performance of the ATC-TA system for the spike protein of wild-type SARS-CoV-2 to detect the spike protein from SARS-CoV-2 variants. E. The general workflow to detect contrived samples using ATC-TA. F. Fluorescence spectra were made by adding spike protein of SARS-CoV-2 to human serum. All the P values were determined using paired t-test. * P＜0.05. Error bars were shown as means ± S.D., n = 3, triplicate.