The "Bloodless" Blood Test: Intradermal Prick Nanoelectronics for the Blood Extraction-Free Multiplex Detection of Protein Biomarkers

Abstract

Protein biomarkers' detection is of utmost importance for preventive medicine and early detection of illnesses. Today, their detection relies entirely on clinical tests consisting of painful, invasive extraction of large volumes of venous blood; time-consuming postextraction sample manipulation procedures; and mostly label-based complex detection approaches. Here, we report on a point-of-care (POC) diagnosis paradigm based on the application of intradermal finger prick-based electronic nanosensors arrays for protein biomarkers' direct detection and quantification down to the sub-pM range, without the need for blood extraction and sample manipulation steps. The nanobioelectronic array performs biomarker sensing by a rapid intradermal prick-based sampling of proteins biomarkers directly from the capillary blood pool accumulating at the site of the microneedle puncture, requiring only 2 min and less than one microliter of a blood sample for a complete analysis. A 1 mm long microneedle element was optimal in allowing for pain-free dermal sampling with a 100% success rate of reaching and rupturing dermis capillaries. Current common micromachining processes and top-down fabrication techniques allow the nanobioelectronic sensor arrays to provide accurate and reliable clinical diagnostic results using multiple sensing elements in each microneedle and all-in-one direct and label-free multiplex biomarkers detection. Preliminary successful clinical studies performed on human volunteers demonstrated the ability of our intradermal, in-skin, blood extraction-free detection platform to accurately detect protein biomarkers as a plausible POC detection for future replacement of today's invasive clinical blood tests. This approach can be readily extended in the future to detect other clinically relevant circulating biomarkers, such as miRNAs, free-DNAs, exosomes, and small metabolites.

Keywords: Biomarkers; Biomolecules; Detection; Nanobioelectronics; Nanosensors.

Figure 1

Fabrication and characterization of the SiNW-FET-based microneedle array sensor. (a) Schematic illustration of the top-down fabrication process. (b) SEM images of the fabricated needles; the needles are 150 μm in width and ca. 250 μm in thickness (scale bar: 125 μm). The green inset shows the opening of the SU-8 layer that forms the device window (scale bar: 25 μm). The blue inset shows a close-up image on one of the two devices inside the device window. The source-drain pads lie on pads fabricated from the device layer for better contact and surface area. The nanowires are a part of the device layer, laying on the buried oxide, and are 125 nm in width and 75 nm high (scale bar: 5 μm). (c) Electrical characterization of a representative device. The source-drain voltage was swept between −0.4 and 0.15 V, and the gate was kept constant at −0.3 V (black curve), −0.2 V (red curve), −0.1 V (green curve), 0 V (blue curve), and 0.1 V (light blue curve). Inset illustrates how the measurement was made, mimicking the ex vivo experiments as close as possible. (d) Transconductance measurements of five individual devices on the same microneedle FET. Vsd was kept constant on 0.1 V while the gate was swept between (−0.3) V to 0.4 V.

Figure 2

Surface modification process. (a) Illustration of modification. Top: microneedles dipped in 150–200 μL, bottom: each needle drop-cast using a microspotter. (b) Schematic illustration of the chemical modification process, with XPS results of different stages of the modification. (c) Schematic illustration of GFP binding to its antibody to test the modification process. (d) Fluorescence microscopy images results of GFP binding to needle after chemical immobilization of GFP-antibody. The needle is shown before (top) and after (bottom) soaking for 10 min in 60 nM GFP. (e) Fluorescence microscopy images results of GFP binding to bare needle. The needle is shown before (top) and after (bottom) soaking for 10 min in 60 nM GFP. (f) Fluorescence microscopy images of Alexa488 chemically immobilized to needles without SU-8 window before (top) and after (bottom) insertion to PDMS. (g) Fluorescence intensity of needles modified before (black) and after (red) soaking for 10 min in 60 nM GFP. Top: with GFP-antibody, bottom: bare needle, respectively, correlating to (d) and (e). (h) Fluorescence intensity of Alexa488 chemically immobilized to needles before (black) and after (red) insertion to PDMS. Without (top) and with SU-8 window (bottom).

Figure 3

Microneedle array dimensions and blood contact. (a) Optical image of comparison in size between common 27G needle for venous blood extraction and the proposed microneedle array sensors. Two types of fabricated microneedles length are shown −400 μm and 1 mm. Scale bar: 5 mm. (b) Schematic illustration of microneedle insertion to the forearm. 1 mm microneedle should reach the blood capillaries in the dermis, while 400 μm needles will not reach as effectively. (c) Optical images showing the microneedle before (top) and after (bottom) a blood droplet was placed on the microneedle. Orange inset shows blood is clearly able to enter the SU-8 window. (d) Optical image showing the microneedle after insertion to the skin. (e) Schematic illustration of a different possible location for protein detection in the blood. The microneedle array can be used in the finger without or with prior pricking.

Figure 4

Skin insertion and contact with blood vessels. (a) Images of the insertion process of the microneedle array sensors into the forearm. (b) Images showing several insertion experiments of 1 mm microneedle array (left) and 0.4 mm microneedle array (right) to the forearm. (c) Summarized results of blood drawing from insertion experiments of 1 mm microneedle array (red) and 0.4 mm microneedle array (green) to the forearm. (d) Statistical distribution of blood drawing success percentage from 50 insertion experiments of 1 mm microneedle array (black) and 0.4 mm microneedle array (red) to the forearm.

Figure 5

In vivo results of different measurements using the microneedle array. (a) Stabilization curves of PSA association at 10 pM (red curve) and 100 pM (blue curve) spiked-PBS solutions. The results indicate that the sensor requires approximately 60 s to achieve a differentiable signal. (b) One cycle close-up view taken once dissociation stabilization is achieved for PSA-spiked PBS buffer. (c) One cycle close-up view taken once dissociation stabilization is achieved for PSA-spiked serum. (d) Normalized response linear curves derived from (a) and (b). The dissociation phase was conducted in 5% EG in 100 μM phosphate buffer solution. Normalized reaction is in comparison to nonspiked buffer or serum, respectively. Pink data relates to nonspecific normalized response to 22 ng/mL cTnI and 21 ng/mL GFP. (e) In vivo intradermal capillary PSA concentrations were measured in four subjects using the microneedle array (green bars) compared to ELISA measurements of PSA concentration in venous blood (blue dots). (f) Multiplex experiment results of normalized response to PSA-spiked buffer from a device modified with PSA-specific antibody (αPSA, black curve) and a device modified with cTnI-specific antibody (αcTnI, red curve). (g) Multiplex experiment results of normalized response to cTnI-spiked buffer from a device modified with PSA-specific antibody (αPSA, black curve) and a device modified with cTnI-specific antibody (αcTnI, red curve). (i) Top: Deviation measurements performed on a single device via multiple entries to (100 pM spiked serum solution). Bottom: Variance measurements were performed between different devices via normalized response against a 100 pM spiked serum.

Figure 6

Laboratory-scale 3D-printed mount. (a) Illustration of the mount that enables the microneedle-based sensor operation. The mount chip is held for the user to prick his finger using the microneedles, followed by capping of the mount and consecutive washing in the appropriate buffer solution. (b) Optical images show the 3D-printed mount and the laboratory-scale system. (c) Optical image illustrating finger pricking using the 3D-printed mount.