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6CCVD Research

Graphene FET Sensors for Alzheimer’s Disease Protein Biomarker Clusterin Detection

At a Glance

Section titled “At a Glance”
MetadataDetails
Publication Date2021-03-26
JournalFrontiers in Molecular Biosciences
AuthorsTheodore Bungon, Carrie Haslam, Samar Damiati, Benjamin O’Driscoll, Toby Whitley
InstitutionsScience for Life Laboratory, Diamond Light Source
Citations45
AnalysisFull AI Review Included

Executive Summary

Section titled “Executive Summary”

This research details the fabrication and characterization of Graphene Field-Effect Transistor (GFET) biosensors optimized for detecting Clusterin, a critical protein biomarker for Alzheimer’s Disease (AD).

  • Ultra-Low Detection Limit: The GFET biosensors achieved an ultra-low Limit of Detection (LoD) of approximately 300 fg/mL (4 fM) for Clusterin using DC 4-probe electrical resistance (4-PER) measurements.
  • High Sensitivity: Detection demonstrated high sensitivity, showing a significant ~118% increase in resistance upon the binding of 1 pg/mL Clusterin antigen.
  • Fabrication Optimization: Thermal annealing (215°C) was implemented post-fabrication, resulting in a 31% resistance reduction and a 43% increase in carrier mobility (from 460 to 660 cm2/Vs), improving baseline sensor performance.
  • Functionalization Strategy: The monolayer CVD graphene surface was functionalized using 1-pyrenebutanoic acid succinimidyl ester (Pyr-NHS) linkers, which bind strongly via non-covalent π-π interactions, followed by anti-Clusterin antibody immobilization.
  • Specificity Confirmed: Specificity was validated against human chorionic gonadotropin (hCG), where a three-orders-of-magnitude higher concentration (100 ng/mL) resulted in only a minor resistance change (-6 ± 3%).
  • Platform Genericity: The developed GFET architecture is generic and shows potential for broad application in detecting biomarkers for other diseases, including Parkinson’s, cancer, and cardiovascular conditions.

Technical Specifications

Section titled “Technical Specifications”
ParameterValueUnitContext
Limit of Detection (LoD)300fg/mLClusterin detection (4-PER)
Molar LoD4fMClusterin detection (4-PER)
Graphene TypeMonolayerN/ACVD grown on 300 nm Si/SiO2
GFET Channel Length (Symmetric)400µmDevice geometry
GFET Channel Width80µmDevice geometry
Annealing Temperature215°CPost-fabrication optimization
Annealing Time30minPost-fabrication optimization
Resistance Reduction (Annealing)31%Bare to Annealed stage
Carrier Mobility Increase (Annealing)43%Bare to Annealed stage (460 to 660 cm2/Vs)
Resistance Change (1 pg/mL Clusterin)~118%Relative to BSA blocking stage
Specificity Test AnalytehCGN/AHuman chorionic gonadotropin
Specificity Test Concentration100ng/mL3 orders of magnitude higher than Clusterin
Specificity Resistance Change (hCG)-6 ± 3%Average change relative to BSA stage
Linker Concentration (Pyr-NHS)2mMFunctionalization step
Antibody Concentration (Anti-Clusterin)20µg/mLFunctionalization step
Raman I(2D)/I(G) Ratio (Mean)~1.11N/AConfirms monolayer quality

Key Methodologies

Section titled “Key Methodologies”

The GFET sensors were fabricated using photolithography and metal lift-off techniques on p++ Si/SiO2 substrates, followed by a multi-stage biofunctionalization process.

  1. Substrate and Patterning: Monolayer CVD graphene was transferred onto a 300 nm Si/SiO2 substrate. GFET channels were patterned using photolithography (LoR and positive PR spin-coating at 3000 rpm) and UV exposure (25 s).
  2. Residue Removal: Samples were post-baked at 180°C for 8 min under Deep UV (DUV) light (254 nm) to dissociate PR/PMMA residues and reduce contact resistance.
  3. Graphene Etching: Unprotected graphene was etched via Ar plasma (50 W RF power, 2.5 min, 6 x 10-7 Torr vacuum).
  4. Contact Deposition: Source, drain, and voltage electrodes were formed by thermal evaporation of 5 nm Chromium (Cr) adhesion layer, followed by sputtering 30 nm of Gold (Au).
  5. Thermal Annealing: GFETs were annealed at 215°C for 30 min to improve transport properties by removing water molecules and polymeric residues.
  6. Linker Immobilization: 2 mM Pyr-NHS ester linker molecules were drop-cast and incubated (4°C for 4 h). The pyrenyl group binds strongly to graphene via non-covalent π-π interactions.
  7. Antibody Binding: Anti-Clusterin antibody (20 µg/mL) was applied and incubated, covalently reacting with the succinimidyl ester group of the linker.
  8. Blocking: 0.5% Bovine Serum Albumin (BSA) solution was deposited to block non-specific binding sites, enhancing sensor specificity.
  9. Antigen Detection: Clusterin antigen (1 to 100 pg/mL) was applied and incubated (37°C for 1 h).
  10. Electrical Characterization: Sensor response was quantified using DC 4-probe electrical resistance (4-PER) measurements and back-gated ID-VG transfer curves (sweep range -100 V to +100 V, fixed drain voltage 50 mV).

Commercial Applications

Section titled “Commercial Applications”

The GFET biosensor platform, characterized by its ultra-low LoD and generic fabrication process, is highly relevant for several high-value diagnostic and sensing markets.

  • Early Disease Diagnostics:
    • Alzheimer’s Disease (AD): Immediate application for rapid, early-stage detection and monitoring of Clusterin and other AD biomarkers (Aβ, Tau).
    • Parkinson’s Disease: Potential for adaptation to detect relevant protein biomarkers for early diagnosis of Parkinson’s.
  • Oncology and Cancer Screening:
    • The generic platform can be functionalized for high-sensitivity detection of cancer biomarkers, demonstrated by the specificity test against hCG (a cancer risk biomarker).
  • Cardiovascular Monitoring:
    • Applicable for detecting cardiac troponin and other protein biomarkers associated with cardiovascular disease and acute myocardial infarction.
  • Point-of-Care (POC) Testing Devices:
    • The GFET architecture offers high signal-to-noise ratio, low energy consumption, and ease of integration with existing electronic systems, making it ideal for developing portable, non-invasive diagnostic tools.
  • DNA and Molecular Diagnostics:
    • The high surface-to-volume ratio and electrical sensitivity of graphene enable label-free detection of DNA hybridization and other molecular diagnostic targets.
View Original Abstract

We report on the fabrication and characterisation of graphene field-effect transistor (GFET) biosensors for the detection of Clusterin, a prominent protein biomarker of Alzheimer’s disease (AD). The GFET sensors were fabricated on Si/SiO 2 substrate using photolithographic patterning and metal lift-off techniques with evaporated chromium and sputtered gold contacts. Raman Spectroscopy was performed on the devices to determine the quality of the graphene. The GFETs were annealed to improve their performance before the channels were functionalized by immobilising the graphene surface with linker molecules and anti-Clusterin antibodies. Concentration of linker molecules was also independently verified by absorption spectroscopy using the highly collimated micro-beam light of Diamond B23 beamline. The detection was achieved through the binding reaction between the antibody and varying concentrations of Clusterin antigen from 1 to 100 pg/mL, as well as specificity tests using human chorionic gonadotropin (hCG), a glycoprotein risk biomarker of certain cancers. The GFETs were characterized using direct current (DC) 4-probe electrical resistance (4-PER) measurements, which demonstrated a limit of detection of the biosensors to be ∼ 300 fg/mL (4 fM). Comparison with back-gated Dirac voltage shifts with varying concentration of Clusterin show 4-PER measurements to be more accurate, at present, and point to a requirement for further optimisation of the fabrication processes for our next generation of GFET sensors. Thus, we have successfully fabricated a promising set of GFET biosensors for the detection of Clusterin protein biomarker. The developed GFET biosensors are entirely generic and also have the potential to be applied to a variety of other disease detection applications such as Parkinson’s, cancer, and cardiovascular.

Tech Support

Section titled “Tech Support”

Original Source

Section titled “Original Source”

References

Section titled “References”
  1. 2016 - “Transport conductivity of graphene at RF and microwave frequencies. [Crossref]
  2. 2011 - Label-free detection of DNA hybridization using pyrene-functionalized single-walled carbon nanotubes: effect of chemical structures of pyrene molecules on DNA sensing performance. [Crossref]
  3. 2008 - Superior thermal conductivity of single-layer graphene. [Crossref]
  4. 2012 - Graphene photonics, plasmonics, and broadband optoelectronic devices. [Crossref]
  5. 2008 - Graphene-based liquid crystal device. [Crossref]
  6. 2017 - “Mapping the electrical properties of large-area graphene. [Crossref]
  7. 2008 - Ultrahigh electron mobility in suspended graphene. [Crossref]

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