Electronic Supplementary Material
Aptamer-based Impedimetric Determination of the Human Blood Clotting Factor IX in Serum using an Interdigitated Electrode Modified with a ZnO Nanolayer
Ong Chong Cheen1,2, Subash C.B. Gopinath1,3*, Veeradasan Perumal1, M.K. Md Arshad1, Thangavel Lakshmipriya1, Yeng Chen4,5, R. Haarindraprasad1, Balakrishnan S. Rao1, Uda Hashim1, Kannaiyan Pandian6
1Institute of Nano Electronic Engineering (INEE)
2Faculty of Engineering Technology, Universiti Malaysia Perlis, 01000 Kangar, Perlis, Malaysia
3School of Bioprocess Engineering, Universiti Malaysia Perlis,
02600 Arau, Perlis, Malaysia
4Department of Oral Biology & Craniofacial Sciences
5Oral Cancer Research and Coordinating Center (OCRCC), Faculty of Dentistry, University of Malaya, 50603, Kuala Lumpur, Malaysia
6Department of Inorganic Chemistry, University of Madras, Guindy Campus, Chennai 600 001, Tamil Nadu, India
*Correspondence to: Dr. Subash C.B. Gopinath, Email:
Materials and Methods
Methods involved in the structural analyses described below were followed from previous literatures [11,13].
Structural characterization by X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS)
To study the crystal quality, morphology, size, and plane orientation, XRD analysis was carried out. The diffractogram was showing pure ZnO (figure S2a). The diffraction peaks corresponding to ZnO were matched with the reference spectra of ZnO JCPDS Card No. 36-1451 and bulk Au JCPDS Card No. 65-2879. The structure can be classified as the wurtzite phase based on the obtained XRD patterns have reflection peaks at 31.86° (100), 34.49° (002), 36.34° (101), 47.63° (102), 56.65° (110), 62.96° (103) and 68.04° (112). The result obtained shows that the pure hexagonal wurtzite ZnO structures have been synthesized with high-quality crystals and c-axis alignment. To further investigate the elemental composition present on the surface of the IDE, XPS study was performed. The binding energies were calibrated within an accuracy of 0.1 eV using C1s (284.6). The Zn 2p XPS spectra consist of two peaks of Zn 2p3/2 and Zn 2p1/2 that correlates to binding energy peaks at 1022.63 and 1045.83, attributed to Zn2+ valance state shown in y figure S2b.
Immobilization confirmation of anti-FIX-antibody through Fourier Transform-Infrared (FTIR) spectroscopy
To investigate the structure of the compound and its vibrational characteristics present on outmost layer of ZnO upon immobilization of anti-FIX-antibody, FTIR spectroscopy analysis was performed. Supplementary figure S2c (i-iii) shows the FTIR spectra of ZnO-APTES, ZnO-APTES-Glutaraldehyde and ZnO-APTES-Glutaraldehyde-antibody immobilizations, respectively. The identification of the various bonds in functional groups of molecules via bending and stretching was assisted by transmission of infrared wave from FTIR. Supplementary figure S2c(i) APTES modification on IDE shows N-H bend type of vibration because the peak lies in between the adsorption range of 1560 and 1640 cm-1. When glutaraldehyde was added on APTES-modified surface, peak shifted into the range of 1625-1750 cm-1 showing the presence of C=O stretch type of vibration presence and thus prove aldehyde-activated surface was generated as illustrated by supplementary figure S2c(ii). Upon immobilization of anti-FIX-antibody on the surface, the peak shifted back to N-H bend vibration type range is showing the presence of amine-binding site for FIX to attach on it as shown in supplementary figure S2c(iii).
Figure S1: FESEM image of low magnification revealing ZnO on surface of IDE.
Figure S2: (a) X-ray diffraction of ZnO surface (b) Survey scan of XPS core level spectra showing binding energy of Zinc, Zn 2p (c) FTIR spectroscopy analysis. (i-iii) shows the FTIR spectra of ZnO-APTES, ZnO-APTES-Glutaraldehyde (GTD) and ZnO-APTES-Glutaraldehyde-antibody immobilizations, respectively.
Figure S3: Anti-factor IX aptamer. Important binding bases are indicated in loop 1 (L1).