Development of a dual-aptamer-based multiplex protein biosensor

Abstract

Parallel biosensors for proteins are becoming more essential for the thorough and systematic investigation of complex biological processes. These tools also enable improved clinical diagnoses relative to single-protein analyses due to their greater information content. If implemented correctly, affinity-based techniques can provide unique advantages in terms of sensitivity and flexibility. Aptamers are increasingly being used as the affinity reagents of choice for protein biosensing applications. Here, we describe the development and characterization of an aptamer-based method for parallel protein analyses that relies on recognition of the target protein by two unique aptamers targeting different epitopes on the protein. Our results show that the technique achieved simultaneous and quantitative detection of thrombin and platelet-derived growth factor-BB (PDGF-BB) with high specificity both in buffered solutions and in serum samples.

Fig. 1. Dual-aptamer detection schematic

Thrombin and PDGF-BB are represented by triangles and double-ellipses, respectively. The sensing aptamers (MB-T, MB-P) containing thrombin aptamer (Apt 1A) and PDGF-BB aptamer (Apt 2A) are each labeled with an MB tag (MB1 and MB2) and flanked by a pair of universal primers (5′p and 3′p). Capture aptamers (Bio-T and Bio-P) containing thrombin aptamer (Apt 1B) and PDGF-BB aptamer (Apt 2B) are biotinylated to be attached to streptavidin-coated magnetic beads. Oligo(dT)s are used as the spacer between the aptamer sequences and the beads. After separation, eluted sensing aptamers are amplified by multiplex PCR and quantified.

Fig. 2. Multiplex detection of proteins using electrophoresis as the readout method

Control or target protein samples were added to binding buffer containing 250 nM MB-T, 500 nM Bio-T, 50 nM MB-P and 500 nM Bio-P. After binding and separation, eluted sensing aptamers were amplified with universal primers for 20 cycles. PCR products were separated by acrylamide gel and detected by gel staining. Higher bands are the products from amplification of the PDGF-BB and thrombin sensing aptamers. Remaining primers run to the bottom of the gel. Lane 1: Buffer; Lane 2: 333 nM thrombin; Lane 3: 333 nM PDGF-BB; Lanes 4-6: both thrombin and PDGF-BB at 33, 100 and 333 nM from left to right; Lanes 7-10: haptoglobin, fibrinogen, C-reactive protein and lysozyme each at 333 nM. Image contrast was adjusted to reduce background.

Fig. 3. Multiplex detection using qPCR as the readout method

Both sets of aptamer pairs were added with serial dilutions of protein mixtures containing both thrombin and PDGF-BB. After binding, separation, and elution, eluted sensing aptamers were amplified with qPCR using template specific primers. (* and # denote C_t values significantly lower than background for thrombin and PDGF-BB, respectively (p < 0.01)). Lower detection limits were 330 pM and 3.3 nM for thrombin and PDGF-BB, respectively.

Fig. 4. Specificity of detection in the presence of competing protein

Both pairs of aptamers were added to serial dilutions of PDGF-BB in the absence or presence of thrombin (top) or to serial dilutions of thrombin in the absence or presence of PDGF-BB (bottom). After separation, eluted sensing aptamers were amplified with universal primers and read out using electrophoresis. All four aptamers were used at 500 nM concentration to assess how much cross reactivity occurred at high aptamer concentrations. Little cross-reactivity was seen in either case.

Fig. 5. Correlation of various analyses

Multiplex C_t values derived in buffer or serum samples were plotted against the C_t value derived from singleplex experiments in buffer for thrombin (a) and PDGF-BB (b).

Fig. 5. Correlation of various analyses

Multiplex C_t values derived in buffer or serum samples were plotted against the C_t value derived from singleplex experiments in buffer for thrombin (a) and PDGF-BB (b).