Abstract
The elevation of cytokine levels in body fluids has been associated with numerous health conditions. The detection of these cytokine biomarkers at low concentrations may help clinicians diagnose diseases at an early stage. Here, we report an asymmetric geometry MoS2 diode-based biosensor for rapid, label-free, highly sensitive, and specific detection of tumor necrosis factor-α (TNF-α), a proinflammatory cytokine. This sensor is functionalized with TNF-α binding aptamers to detect TNF-α at concentrations as low as 10 fM, well below the typical concentrations found in healthy blood. Interactions between aptamers and TNF-α at the sensor surface induce a change in surface energy that alters the current-voltage rectification behavior of the MoS2 diode, which can be read out using a two-electrode configuration. The key advantages of this diode sensor are the simple fabrication process and electrical readout, and therefore, the potential to be applied in a rapid and easy-to-use, point-of-care, diagnostic tool.
Introduction
Cytokines are small proteins that play an important role in regulating the inflammatory response. Found in biofluids such as blood, saliva, and sweat, they have gained interest as biomarkers for various health conditions and diseases1,2,3. An abnormal change in cytokine concentration is an indicator of uncontrolled inflammatory reactions that has been linked to Alzheimer’s disease, cancers, pulmonary tuberculosis, autoimmune, and cardiovascular disease1,4,5,6,7. In addition, coronavirus 2019 (COVID-19) infection is accompanied by a release of an elevated level of pro-inflammatory cytokines such as interleukins (IL-1β and IL-6) and tumor necrosis factor-α (TNF-α), in an occurrence called a cytokine storm8. Studies have suggested that cytokine inhibitors are an effective treatment for improving COVID-19 survival9. Treatment of many diseases is most effective at an early stage10. Thus, the ability to monitor and detect early changes in inflammatory cytokine levels is of great interest to clinical diagnosis. Serum levels of TNF-α among healthy young and adult population is typically in the range of 200–300 fM11. In the case of children, the serum levels can be as low as 12 fM12. Hence, having a limit of detection (LOD) in the range of fM is important in early diagnostic applications.
The standard method for measuring cytokines is via an enzyme-linked immunosorbent assay (ELISA), a technique which is widely used in clinical laboratories and biomedical research13. Single molecule array (SIMOA), an ultrasensitive ELISA method, and mass spectroscopy can detect cytokines at concentrations in the fM range, sufficiently sensitive to monitor diseases in an individual14. However, these methods are time-consuming and expensive, limiting widespread use for diagnostic applications.
Biosensors are analytical devices that consist of a biorecognition element (the receptor) on a transducer, which transforms the interactions between the biorecognition element and the specific target into a measurable signal15. There are a number of different sensing mechanisms in biosensors, including optical, electrical, acoustic and electrochemical.16,17. For example, Ghosh S et al. reported detection of TNF-α using a quantum dot-based optical aptasensor with a LOD in the pM range18. A malaria biomarker employing an antibody-aptamer plasmonic biosensor reported a LOD of 18 fM16.
Among the different types of biosensors, field-effect transistor (FET) based biosensors have attracted great attention by providing a label-free, rapid, low power consumption, portable, and highly sensitive sensing platform that can be easily integrated with other electronic components such as data analyzers and signal transducer applications2. However, FET biosensors, including graphene-based FETs (GFETs) and FETs based on transition metal dichalcogenides (TMDs), are generally used in a liquid-gated configuration with an Ag/AgCl electrode, which can hinder the integration and scaledown of the device2. The gate electric field, applied via a sample solution, can disturb the binding affinity between the charged cytokines and receptors, hence affecting the sensing stability19. Also, the continuous electrical stress in the liquid can lead to undesirable leakage current, that would generate false sensor response and electronically damage the sensor20. A serious issue related to TMD-based FET sensors is the hysteresis in the transfer characteristics arising due to gate-modulated charges trapped at the TMD/dielectric interfaces. This behavior makes the current measured across the drain and source, under a given gate voltage, highly dependent on the sweep range, sweep direction, sweep time and loading history of gate voltage biases, which leads to inconsistent sensor readings20. A solid-gated FET employing a dielectric layer could mitigate some of the issues present in the liquid-gate sensors. However, the solid-gate FETs, particularly devices with thick SiO2 dielectric layer, typically require high operating gate voltages in the range of 40–50 V for GFETs and ∼100 V for TMD-based FETs20, which can be a human health hazard2,21.
By employing a simple two-electrode diode sensor, issues associated with gating can be completely avoided. A number of recent publications have reported the diode rectification behavior arising due to an asymmetry in contact geometries for several 2D-materials including graphene, WSe2,WS2, In2S3, and GeAs22,23,24,25,26,27, and also in a varying diameter Si nanowire (NW)28. An asymmetric contact geometry also offers a simple fabrication process requiring only one metallization layer (Cr/Au) and no doping process.
Here, we report the detection of the pro-inflammatory cytokine TNF-α, at concentrations as low as 10 fM using an asymmetric geometry diode, with a wide dynamic range of detection (5 orders of magnitude) from concentrations of 10 fM to 1 nM. We employ a well-known silane functionalization process and aptamer cytokine interaction to demonstrate the detection of cytokines using this device architecture. The device’s rectification factor (RF), the ratio of current measured at reverse and forward bias voltages (−1 and +1 V), changes as a function of cytokine concentration, when added to the sensor surface in a phosphate-buffer saline solution (PBS). The ultra-sensitivity, we presume is due to the change in charge density at the surface of the TMDs, generated by cytokine-aptamer interactions. Our biosensor offers several key advantages compared to existing methods of cytokine measurement including a smaller sample volume, (3 μl of TNF-α diluted in PBS compared to the typical volume of 100 μl used for ELISA), rapid measurement without the need for incubation, minimal hands-on time, and no requirement for expensive optical equipment. Moreover, the diode sensor uses only two electrodes, which simplifies the fabrication process and readout electronics compared to field-effect-transistors (FETs), which require a third gate electrode. Due to its simple operation, and absence of complicated post-measurement analysis, the diode sensor requires minimal training and is therefore suitable for point-of-care diagnostic applications. Since the detection of TNF-α inherently depends upon the bioreceptor (aptamer) anchored to the sensing area, the proposed sensor application can be extended to detect other cytokines, proteins, or other biomarkers molecules by replacing the bioreceptor with one that specifically binds to the corresponding biomarker. Due to the flexibility and mechanical stability of 2D MoS2, a flexible and wearable biosensor device for health monitoring applications is feasible. Considering these advantages, we anticipate that our cytokine biosensor has the potential for application as an easy-to-use, point-of-care biomarker testing tool.
Results
Aptamer-based cytokine diode sensor
An illustration of the cytokine measurement procedure using an asymmetric geometry MoS2 diode is shown in Fig. 1. Note that the blood sample shown in Fig. 1a is a conceptual illustration of how TNF- α concentrations would be measured in a blood sample. The sensor consists of a 2D semiconductor, (2H-phase) multilayer MoS2 crystal flake on top of thermally-oxidized SiO2 (oxide thickness of 300 nm) contacted by two Cr/Au electrodes. Based on atomic force micrscopy (AFM) measurements, the typical MoS2 thicknesses were between 13 and 60 nm (Supplementary Fig. 1). Cr/Au contacts were fabricated with photolithography to form two electrical contacts across the flake, with an electrode spacing of 10 µm. The geometric asymmetry of the MoS2 crystal, with two MoS2-metal contacts of different cross-sectional lengths and areas, induces a diode rectification behavior. The MoS2 flake is coated with a thin insulating Al2O3 layer, which is then functionalized with an aptamer that specifically binds to the targeted cytokine TNF-α. When a sample containing TNF-α (e.g., the blood serum in Fig. 1a) is drop cast onto the sensor, the TNF-α cytokines bind to the aptamer forming a G-quadruplex structure that leads to negatively-charged TNF-α moving closer to the sensor surface. Changes in surface charge density induces a change in the electrical rectification behavior in the asymmetric geometry MoS2 diode.
