Nanostructuring of biosensing electrodes with nanodiamonds for antibody immobilization

Abstract

While chemical vapor deposition of diamond films is currently cost prohibitive for biosensor construction, in this paper, we show that sonication-assisted nanostructuring of biosensing electrodes with nanodiamonds (NDs) allows harnessing the hydrolytic stability of the diamond biofunctionalization chemistry for real-time continuous sensing, while improving the detector sensitivity and stability. We find that the higher surface coverages were important for improved bacterial capture and can be achieved through proper choice of solvent, ND concentration, and seeding time. A mixture of methanol and dimethyl sulfoxide provides the highest surface coverage (33.6 ± 3.4%) for the NDs with positive zeta-potential, compared to dilutions of dimethyl sulfoxide with acetone, ethanol, isopropyl alcohol, or water. Through impedance spectroscopy of ND-seeded interdigitated electrodes (IDEs), we found that the ND seeds serve as electrically conductive islands only a few nanometers apart. Also we show that the seeded NDs are amply hydrogenated to be decorated with antibodies using the UV-alkene chemistry, and higher bacterial captures can be obtained compared to our previously reported work with diamond films. When sensing bacteria from 10(6) cfu/mL E. coli O157:H7, the resistance to charge transfer at the IDEs decreased by ∼ 38.8%, which is nearly 1.5 times better than that reported previously using redox probes. Further in the case of 10(8) cfu/mL E. coli O157:H7, the charge transfer resistance changed by ∼ 46%, which is similar to the magnitude of improvement reported using magnetic nanoparticle-based sample enrichment prior to impedance detection. Thus ND seeding allows impedance biosensing in low conductivity solutions with competitive sensitivity.

Figure 1

Measurement of (A) ND particle sizes, (B) mobility (μ_e), and (C) zeta-potential (ζ) after dilution of the original ND-containing DMSO with acetone, ethanol, isopropyl alcohol (IPA), methanol, and water. Average values are reported from five repeats. (D) On the left is the SEM image showing NDs (bright white spots) seeded on gold surfaces by sonication in solution containing NDs at 0.25% (w/v) for 30 min. On the right is the SEM image after 2D FFT filtering highlights the seeded NDs as red regions. (E) Surface coverage obtained on gold surfaces by sonication in ND solution diluted with acetone, ethanol, IPA, methanol, and water. (F) Surface coverage of NDs obtained on surfaces seeded for varying amounts of times with methanol solutions containing 0.25, 0.125, and 0.08% (w/v) NDs.

Figure 2

Fluorescence images obtained from a 10 × 10 array of 12 μm spots of FITC-labeled anti-E. coliO+K attached to ND-seeded surfaces without (A) and with (B) sodium borohydride reduction treatment prior to performing UV-assisted TFAAD linkage to NDs. (C) Normalized fluorescence intensity obtained from seven 10 × 10 arrays on the ND surface with and without reduction.

Figure 3

Bacteria capture density obtained using the antibody-ND coating as a function of surface coverage of NDs on the sample.

Figure 4

(A) Optical images of a biosensor chip containing an array of nine interdigitated electrode (IDE) pairs that were fabricated to demonstrate the application of ND seeding layer for chemically stable covalent linkage of antibodies to electrodes. Each IDE contained 60 finger pairs with each finger 9 μm wide and spaced 9 μm apart. (B) Representative plot of real versus imaginary part of the impedance measured in deionized water on an IDE before and after ND seeding. (C) Representative plot of impedance magnitude and phase plotted against frequency as obtained on an IDE exposed to solutions with different electrical conductivity.

Figure 5

(A) Example impedance spectra on active sensors before (baseline) and after exposure to 10⁶ or 10⁸ cells/mL of E. coliO157:H7 cells. (B) Modified Randles circuit that best fits our impedance results. (C) Significant changes observed in charge transfer resistance (R_dl, R_fl, and Ws1-R) upon binding of bacterial cells to the sensor surface.