Highly efficient bienzyme functionalized nanocomposite-based microfluidics biosensor platform for biomedical application

Abstract

This report describes the fabrication of a novel microfluidics nanobiochip based on a composite comprising of nickel oxide nanoparticles (nNiO) and multiwalled carbon nanotubes (MWCNTs), as well as the chip's use in a biomedical application. This nanocomposite was integrated with polydimethylsiloxane (PDMS) microchannels, which were constructed using the photolithographic technique. A structural and morphological characterization of the fabricated microfluidics chip, which was functionalized with a bienzyme containing cholesterol oxidase (ChOx) and cholesterol esterase (ChEt), was accomplished using X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy. The XPS studies revealed that 9.3% of the carboxyl (COOH) groups present in the nNiO-MWCNT composite are used to form amide bonds with the NH2 groups of the bienzyme. The response studies on this nanobiochip reveal good reproducibility and selectivity, and a high sensitivity of 2.2 mA/mM/cm2. This integrated microfluidics biochip provides a promising low-cost platform for the rapid detection of biomolecules using minute samples.

Figure 1

(i) The schematic of the microfluidic biochip used for total cholesterol detection (the ordered arrangement of this microsystem is assumed). (ii) The photograph of real microfluidic biochip for cholesterol detection and (iii) the enlarged view of optical microscopic image of the microfluidic biochip.

Figure 2

(i) The X-ray diffraction pattern of nNiO-MWCNTs and (ii) the Raman spectroscopy spectra of the NiO nanoparticles (a) and nNiO-MWCNTs (b).

Figure 3

(i) The wide-scan X-ray photoelectron spectra (XPS) of various films. (ii) The XPS spectra of the C1s region of the nNiO-MWCNT film after deconvolution; (iii) the C1s region of the ChEt-ChOx/nNiO-MWCNT/ITO film; and (iv) the N1s core-level spectra of the ChEt-ChOx/nNiO-MWCNT/ITO film.

Figure 4

The HR-TEM analysis of (i) the NiO nanoparticles, (ii) an individual MWCNT (inset: the SAED pattern of the MWCNT), (iii) the MWCNTs modified with NiO nanoparticles, and (iv) an atomic-scale image of a nNiO-MWCNT (inset: lattice fringes of the MWCNT).

Figure 5

(A) A cyclic voltammogram (CV) of the different electrodes in a PBS solution (50 mM, pH 7.0, 0.9% NaCl) containing 5 mM of [Fe(CN)₆]^3−/4− (inset: current versus flow rate plot of the chronoamperometric response). (B) The electrochemical impedance spectroscopy (EIS) spectra of the electrodes (inset: a schematic representation of the Randles equivalent circuit model for impedance measurement).

Figure 6

(i) The chronoamperometric response of the ChEt-ChOx/nNiO-MWCNT/ITO-based biochip as a function of the cholesterol oleate concentration (0.25–12.93 mM) in a PBS solution containing 5 mM of [Fe(CN)₆]^3−/4−. The experiment was controlled using a syringe pump attached to the inlet of the microsystem (inset: the response current as a function of the cholesterol concentration obtained for both (a) ChEt-ChOx/nNiO-MWCNT/ITO and (b) nNiO-MWCNT/ITO electrodes). (ii) A calibration plot showing the logarithm of the cholesterol concentration (mM) and the amperometric current of the biochip during sensing.

Figure 7

(i) Reproducibility studies of the ChEt-ChOx/nNiO-MWCNT/ITO microfluidics bioelectrodes under similar conditions. (ii) Stability studies of the ChOx-ChEt/nNiO-MWCNT/ITO bioelectrode and (iii) selectivity studies of the ChOx-ChEt/nNiO-MWCNT/ITO-based biochip.