Quantification of cDNA on GMR biosensor array towards point-of-care gene expression analysis

Abstract

Gene expression analysis at the point-of-care is important for rapid disease diagnosis, but traditional techniques are limited by multiplexing capabilities, bulky equipment, and cost. We present a gene expression analysis platform using a giant magnetoresistive (GMR) biosensor array, which allows multiplexed transcript detection and quantification through cost-effective magnetic detection. In this work, we have characterized the sensitivity, dynamic range, and quantification accuracy of Polymerase chain reaction (PCR)-amplified complementary DNA (cDNA) on the GMR for the reference gene GAPDH. A synthetic GAPDH single-stranded DNA (ssDNA) standard was used to calibrate the detection, and ssDNA dilutions were qPCR-amplified to obtain a standard curve. We demonstrate that the GMR platform provides a dynamic range of 4 orders of magnitude and a limit of detection of 1 pM and 0.1 pM respectively for 15 and 18-cycle amplified synthetic GAPDH PCR products. The quantitative results of GMR analysis of cell-line RNA were confirmed by qPCR.

Keywords: Array; DNA melting; GMR sensors; Gene expression; PCR; Point-of-care.

Figure 1.

A) Target DNA preparation. HeLa mRNA was reverse transcribed and PCR-amplified to cDNA with biotinylated GAPDH primers. PCR product was denatured in 95°C to create target ssDNA. B) Sensors on the GMR chip are spotted with a GAPDH, negative control, and positive control, according to the spotting pattern seen. GAPDH sensor is functionalized with DNA probes complementary to the GAPDH target ssDNA in (A). This target ssDNA is added to the sensor surface and allowed to hybridize. Signal is measured after adding streptavidin MNPs.

Figure 2.

A). 40-cycle amplification curves for both GAPDH standard ssDNA diluted down in a 10x series dilution and HeLa cDNA amplified with GAPDH primers. B) The intensity values (y-axis) from the GAPDH standards in (A) were extracted and plotted for respective cycle numbers (x-axis), and fit to sigmoid functions. Both 15 and 18 cycles gave the largest working range of detection (4 orders of magnitude).

Figure 3.

qPCR standard curve plotted for varying concentrations of synthetic GAPDH DNA. The data were fit to a linear function, with an efficiency of 77.6%, an R² value of 0.994, a slope of −4.007, and a y intercept of −27.594. The average concentration of the HeLa cDNA was determined to be 6.0 ± 0.8 pM after fitting to the standard curve.

Figure 4.

A) GAPDH standard was diluted down pre-PCR 10x in a series dilution (10 nM→ 1 pM) and then PCR amplified for 15 cycles. 0.01 nM is the qPCR limit of detection; 1 pM showed no amplification. B) GAPDH standard was diluted down pre-PCR 10x in a series dilution (10 nM→ 0.1 pM) and then PCR amplified for 18 cycles. 1 pM is the qPCR limit of detection; 0.1 pM showed no amplification. C) 15-cycle PCR product was added to different GMR chips and allowed to hybridize for 1 hour. Data was fit to a semi-logarithmic trendline, showing a working range of 4 orders of magnitude (1 pM→10nM), with a limit of detection of 1 pM. D) 18-cycle PCR product was added to different GMR chips and allowed to hybridize for 1 hour. Data was fit to a semi-logarithmic trendline, showing a working range of 4 orders of magnitude (0.1 pM→1 nM), with a limit of detection of 0.1 pM. The signal recorded at 10 nM represents an outlier; this point is out of the 18-cycle dynamic range. For both (C) and (D), points represents the mean GMR signal saturation level for the 32 GAPDH sensors, normalized to the mean negative control signal. Error bars indicate standard deviation in GAPDH signal between the 32 GAPDH sensors. Background line indicates the GMR signal measured from the no-template control.