Label-free biomarker detection from whole blood

Abstract

Label-free nanosensors can detect disease markers to provide point-of-care diagnosis that is low-cost, rapid, specific and sensitive. However, detecting these biomarkers in physiological fluid samples is difficult because of problems such as biofouling and non-specific binding, and the resulting need to use purified buffers greatly reduces the clinical relevance of these sensors. Here, we overcome this limitation by using distinct components within the sensor to perform purification and detection. A microfluidic purification chip simultaneously captures multiple biomarkers from blood samples and releases them, after washing, into purified buffer for sensing by a silicon nanoribbon detector. This two-stage approach isolates the detector from the complex environment of whole blood, and reduces its minimum required sensitivity by effectively pre-concentrating the biomarkers. We show specific and quantitative detection of two model cancer antigens from a 10 microl sample of whole blood in less than 20 min. This study marks the first use of label-free nanosensors with physiological solutions, positioning this technology for rapid translation to clinical settings.

Figure 1. Schematic of MPC operation

a, Primary antibodies to multiple biomarkers, here prostate specific antigen (PSA) and carbohydrate antigen-15.3 (CA15.3), are bound with a photocleavable crosslinker to the MPC. The chip is placed in a plastic housing and a valve (pink) directs fluid flow exiting the chip to either a waste receptacle or the nanosensor chip. b, Whole blood is injected into the chip with the valve set to the waste compartment (black arrow shows the direction of fluid flow) and, if present in the sample, biomarkers bind their cognate antibodies. c, Washing steps follow blood flow and the chip volume (5 μL) is filled with sensing buffer prior to UV irradiation (orange arrows). During UV exposure, the photolabile crosslinker cleaves, releasing the antibody-antigen complexes into solution. d, The valve is set to the nanosensor reservoir (black arrow shows the direction of fluid flow) and the 5 μL volume is transferred, enabling label-free sensing to be performed to determine the presence of specific biomarkers.

Figure 2. MPC operation

a, Molecular structure of the photocleavable crosslinker. Primary antibody conjugation was performed with the amino group (right) and binding to chip-bound avidin occurred through the biotin group (left). b, Scanning electron micrograph of a representative w = 4 mm × l = 7 mm × h = 100 μm MPC capture-release chip. The inset is an optical image of MPC operation during washing. c, Schematic representation of PSA and CA15.3 detection using a modified ELISA technique. d, Fluorescence optical micrograph of an anti-OVA functionalized MPC following OVA-FITC-spiked whole blood flow and washing. The inset plots the pixel intensity (gray value, determined by ImageJ) versus position for the red cutline (green dataplot) and similar cutlines from images of post-UV irradiation and transfer (blue) and of an anti-PSA functionalized MPC following OVA-FITC-spiked blood flow and washing. The same exposure times were used for all images. e, Scatter plot showing the concentration of PSA and f, CA15.3 released from the MPC versus the concentration of PSA and CA15.3 introduced in whole blood, respectively. Each datapoint represents the average of three separate MPC runs and error bars represent one standard deviation.

Figure 3. Nanosensor electrical characteristics

a, Optical image of devices outfitted with sensing reservoirs. The inset shows an optical micrograph of a completed device. Only the central region of the device (black arrow) is exposed to the solution. Metal leads contact the device source and drain and fan out to larger contacts (not shown). The 25 nm thick silicon device appears yellow. b, I_DS(V_DS) plot for V_G varied from 0 to -20V (black arrow shows direction of increasing negative V_G) for a representative device illustrating p-type accumulation mode behavior. c, I_DS(V_G) plot (V_DS = 1V) for the device used in b. The inset highlights I_DS (nA) around the operating point (V_G = -5V). d, Plot demonstrating the effect of varying solution gate voltage (V_{G, SOLN}) on device current (I_DS; black solid) and device-to-solution leakage current (I_LEAK; blue dashed) for V_DS = 1V.

Figure 4. Label-free sensing

All sensing measurements were performed at V_DS = 1V and V_G = -5V and all sample introductions occurred at time = 0. Normalizations were performed by dividing device currents by the pre-addition (time < 0) current level average. a, Response of an anti-PSA functionalized sensor to a MPC-purified blood sample initially containing 2.5 ng/mL PSA (and also 30 U/mL CA15.3), or a control sample containing neither. b, Response of an anti-CA15.3 functionalized sensor to a MPC-purified blood sample blood sample initially containing 30 U/mL CA15.3 (and also 2.5 ng/mL PSA), or a control sample containing neither. c, Normalized response of two anti-PSA and d, two anti-CA15.3 functionalized devices to MPC-purified blood containing both PSA and CA 15.3, with concentrations labeled. A least squares fit is represented by a solid black line, over the selected region (line endpoints). The ratio of the the normalized slopes calibrates the ratio of concentrations.