A rapid-response ultrasensitive biosensor for influenza virus detection using antibody modified boron-doped diamond
At a Glance
Section titled âAt a Glanceâ| Metadata | Details |
|---|---|
| Publication Date | 2017-11-10 |
| Journal | Scientific Reports |
| Authors | Dawid Nidzworski, Katarzyna Siuzdak, PaweĆ NiedziaĆkowski, Robert Bogdanowicz, MichaĆ Sobaszek |
| Institutions | GdaĆsk University of Technology, Polish Academy of Sciences |
| Citations | 135 |
| Analysis | Full AI Review Included |
6CCVD Technical Analysis: Ultrasensitive Boron-Doped Diamond Biosensors for Influenza Detection
Section titled â6CCVD Technical Analysis: Ultrasensitive Boron-Doped Diamond Biosensors for Influenza DetectionâReference: D. Nidzworski et al. A rapid-response ultrasensitive biosensor for influenza virus detection using antibody modified boron-doped diamond. Sci. Rep. 7, 15707 (2017).
Executive Summary
Section titled âExecutive SummaryâThis research establishes a novel, high-performance electrochemical biosensor utilizing boron-doped polycrystalline diamond (BDD) electrodes for the detection of the universal influenza M1 protein biomarker. The key achievements demonstrate superior performance compared to traditional virological diagnostics.
- Ultra-High Sensitivity: Achieved an unprecedented Limit of Detection (LOD) of 1 fg/ml for the M1 protein in saliva buffer, corresponding to the detection of only 5-10 virus particles per sample.
- Rapid Response Time: The sensor exhibits a response time of less than 5 minutes (incubation), making it significantly faster than standard PCR methods which require hours.
- Universal Applicability: The sensor successfully detects both H1N1 and H3N2 influenza A strains, confirming the approachâs universality based on the target M1 protein.
- Material Excellence: Performance relies on the outstanding electrochemical properties of highly conductive BDD (acceptor concentration 3 × 1021 cm-3), featuring a low background current and wide potential window.
- Validated Selectivity: The biosensor maintains high selectivity and performance even in the presence of common throat infection interfering pathogens (Candida Albicans and Streptococcus Aureus).
- Fabrication Method: The BDD layer was deposited using Microwave Plasma Chemical Vapor Deposition (MPCVD) and subsequently functionalized using a 4-aminobenzoic acid diazonium salt SAM (Self-Assembled Monolayer) for antibody grafting.
Technical Specifications
Section titled âTechnical Specificationsâ| Parameter | Value | Unit | Context |
|---|---|---|---|
| Material Base | Boron-Doped Diamond (BDD) | N/A | Polycrystalline film deposited on Si(100) substrate |
| BDD Film Thickness | ~2 | ”m | Grown over approximately 6 hours via MPCVD |
| Boron/Carbon Ratio | 10,000 | ppm | Diborane (B2H6) doping precursor gas ratio |
| Acceptor Concentration | 3 × 1021 | cm-3 | High conductivity BDD film |
| Detection Mechanism | Electrochemical Impedance Spectroscopy (EIS) | N/A | Measures Rct (Charge Transfer Resistance) change |
| Limit of Detection (LOD) | 1.0 | fg/ml | M1 protein detection in saliva buffer |
| Calculated LOD | 0.7 | fg/ml | Calculated based on 3 × SD/slope |
| Sensor Sensitivity (Linear Range) | 2.7 | kΩ/fg | Linear increase up to 100 fg/ml M1 concentration |
| Minimum Virus Load Detected | 5-10 | viruses/sample | Corresponding to the 1 fg/ml M1 LOD |
| Incubation/Response Time | < 5 | minutes | Measured for H1N1 virus solution (V1) |
| Working Electrode Geometry | 8 | mm diameter | Circular active surface area 0.1256 cm2 |
| Operating Temperature | Room | °C | Electrochemical measurements taken after 1 min stabilization |
| Redox Species | 1 mM K3Fe(CN)6 | N/A | Used in 0.1 PBS electrolyte |
Key Methodologies
Section titled âKey MethodologiesâThe biosensor fabrication involved stringent MPCVD growth conditions followed by a multi-step surface functionalization protocol leveraging the inertness and stability of the BDD surface.
1. MPCVD Diamond Synthesis (BDD Fabrication):
- System: MW PA CVD system (Seki Technotron AX5400 S).
- Substrate Preparation: p-type Si (100) wafers, cleaned (acetone, 2-propanol sonication), and seeded in nanodiamond suspension (crystallite size 5-10 nm).
- Growth Conditions:
- Substrate Temperature: 700 °C.
- Microwave Power: 1300 W (2.45 GHz).
- Gas Mixture: 1% molar ratio CH4 in H2.
- Process Pressure: 50 Torr.
- Doping: Diborane (B2H6) used as dopant precursor, resulting in a [B]/[C] ratio of 10,000 ppm.
2. Surface Pre-treatment and Termination:
- Cleaning/Etching: Substrates received hot aqua regia and hot piranha solution cleaning to remove metallic and organic impurities.
- H-Termination: Microwave hydrogen plasma treatment (1000 W power, 300 sccm flow) for 10 minutes to achieve a predominantly hydrogen-terminated surface.
3. Electrochemical Functionalization (BDD-aM1-BSA):
- Step 1: Diazonium Salt Grafting: BDD surface electrochemically modified using 4-aminobenzoic acid diazonium salt (SAM) by cycling potential from 0 to -1.0 V (10 scans). This creates a continuous monolayer of carboxyl groups.
- Step 2: Antibody Activation: Carboxyl groups activated using EDC/NHS chemistry (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride / N-hydroxysuccinimide).
- Step 3: Antibody Grafting (aM1): Polyclonal anti-M1 antibodies (6 ”g/ml) were captured onto the SAM (24 hours at 4 °C).
- Step 4: Blocking/Saturation (BSA): The BDD-aM1 electrode was saturated with a 0.5% Bovine Serum Albumin (BSA) solution (1 hour at 4 °C) to block non-specific binding sites.
6CCVD Solutions & Capabilities
Section titled â6CCVD Solutions & CapabilitiesâThis research confirms the critical role of highly conductive, high-quality Boron-Doped Diamond (BDD) films in creating ultra-sensitive electrochemical biosensors. Replicating or extending this highly successful platform requires tight control over material synthesis, doping levels, and post-processing capabilitiesâall core strengths of 6CCVD.
Applicable Materials
Section titled âApplicable MaterialsâTo achieve the exceptional electrochemical performance demonstrated in this paper (Acceptor Concentration: 3 × 1021 cm-3), researchers require highly conductive BDD optimized for low background current and maximum stability.
| 6CCVD Material Specification | Application in Biosensor Research |
|---|---|
| Heavy Boron-Doped PCD (Polycrystalline Diamond) | Ideal material foundation. 6CCVD offers PCD with custom doping profiles ([B]/[C] up to 20,000 ppm) to ensure the necessary bulk conductivity and wide potential window for high-sensitivity EIS. |
| Optical/Electronic Grade Substrates | We provide high-quality Si, sapphire, or customized substrates up to 125 mm diameter for subsequent MPCVD growth, matching the experimental substrate requirements. |
| Custom Thickness Control | Researchers used a 2 ”m film. 6CCVD guarantees precise thickness control for PCD layers from 0.1 ”m up to 500 ”m, allowing optimization for sensing, conductivity, and thermal management. |
Customization Potential
Section titled âCustomization PotentialâThe success of the biosensor relies on precise geometry and surface preparation before functionalization. 6CCVDâs in-house capabilities directly support the scaling and optimization of this platform.
- Custom Dimensions and Etching: The paper utilized 8 mm diameter electrodes cut from a larger wafer. 6CCVD offers high-precision laser cutting and patterning to deliver BDD/PCD wafers in highly specific geometries, diameters, and shapes required for microfluidic integration or high-throughput arrays.
- Polishing Services: While the functionalization uses a chemical attachment method, surface roughness affects charge transfer. 6CCVD guarantees state-of-the-art polishing, achieving Ra < 5 nm for inch-size PCD, providing a reliably smooth and consistent starting surface for SAM deposition.
- Integrated Metalization: For robust electrical contacts necessary for EIS instrumentation, 6CCVD offers custom, in-house metalization services. We routinely deposit Au, Pt, Ti, W, and Pd stacks, ensuring low-resistance ohmic contacts compatible with electrochemical measurements.
Engineering Support
Section titled âEngineering SupportâThis research utilized advanced surface chemistry (diazonium salts, EDC/NHS coupling) combined with specific H-termination. 6CCVDâs in-house PhD engineering team specializes in material preparation and surface termination for electrochemical applications.
We offer expert consultation to assist researchers in selecting the optimal BDD doping level, film thickness, and pre-treatment protocol (e.g., oxygen or hydrogen termination) required to replicate this ultra-sensitive influenza biosensor or extend the concept to other applications, such as the detection of universal biomarkers for SARS-CoV-2 or other infectious diseases.
For custom specifications or material consultation, visit 6ccvd.com or contact our engineering team directly.
View Original Abstract
Abstract According to the World Health Organization (WHO), almost 2 billion people each year are infected worldwide with flu-like pathogens including influenza. This is a contagious disease caused by viruses belonging to the family Orthomyxoviridae. Employee absenteeism caused by flu infection costs hundreds of millions of dollars every year. To successfully treat influenza virus infections, detection of the virus during the initial development phase of the infection is critical, when tens to hundreds of virus-associated molecules are present in the patientâs pharynx. In this study, we describe a novel universal diamond biosensor, which enables the specific detection of the virus at ultralow concentrations, even before any clinical symptoms arise. A diamond electrode is surface-functionalized with polyclonal anti-M1 antibodies, which then serve to identify the universal biomarker for the influenza virus, M1 protein. The absorption of the M1 protein onto anti-M1 sites of the electrode change its electrochemical impedance spectra. We achieved a limit of detection of 1 fg/ml in saliva buffer for the M1 biomarker, which corresponds to 5-10 viruses per sample in 5 minutes. Furthermore, the universality of the assay was confirmed by analyzing different strains of influenza A virus.