Cholesterol biosensors have recently gained much attention due to the high rate of clinical disorders, such as heart disease, coronary artery disease, and cerebral thrombosis, among others. The development of a biosensor that integrates cholesterol oxidase for the detection of cholesterol is important as a diagnosis tool. In this investigation, carbon nanofiber electrodes were used for protein immobilization. The frequently used techniques for this procedure are protein: entrapment, physical absorption and covalent bonding. However, covalent bonding that is based on the binding of functional group of the enzymes and matrix via covalent bonds, improves uniformity, density and distribution of the bound protein. This method is being studied for the preparation of the biosensor. Characterization of the biosensor was done using surface and electrochemicalmethodsto guarantee successful immobilization of cholesterol oxidase.

Abstract

Bio-Receptor Transducer

Schematic representation of a biosensor containing the Bio-recognition element (bio receptor) that converts substrate into products, the transducer (that converts the biological recognition into an electrical signal) and the output whose magnitude is related to the magnitude concentration of the analyte of interest.

Cholesterol Oxidase Enzyme

His 447

Glu 361

Asn 485

Cholesterol Oxidase

Active Site

Cholesterol Oxidase Catalyzes the Reaction with Cholesterol:

Molecular mass=55kDa, ~ 4 nm

Acidic = pl 4.4-5.1

Acknowledgements

Future Work

This work was financially supported in part by NASA Center for Advanced Nanoscale Materials under Grant Number NNX08BA48A. DS acknowledge the Harriett Jenkins Pre-Doctoral Fellowship Program (JPFP). Ramsey M. Stevens from Carbon Design Innovations for the CNT Tips.

Cholesterol Oxidase Immobilization on Carbon Nanofiber Electrode for Biosensor ApplicationDámaris Suazo-Davila1a, Johary Rivera1b, Jessica Koehne2, Meyya Meyyappan2, Carlos R. Cabrera1c

(1)Department of Chemistry and NASA-URC Center for Advanced Nanoscale Materials, University of Puerto Rico, Rio Piedras Campus, P.O. Box 70377-San Juan,PR 00936-(a)suazo.damaris@gmail.com, (b) jorime22@gmail.com, (c) carlos.cabrera2@upr.edu (2) NASA Ames Research Center, Moffett Field, California 94035- (a)jessica.e.koehne@nasa.gov,(b)m.meyyappan@nasa.gov

Background Information1

2

3

SEM and AFM

Conclusions This work presents the surface and electrochemical characterization of the CNF electrode. The Scanning Electron Miscroscopy (SEM) and Atomic force microscopy (AFM) images show the different surfaces obtained during the development of the CNF. AFM characterization of the polished CNF electrodes indicated that the CNFs had a mean diameter of 100 nm and protruded 12 nm from the silicon dioxide. X-ray photoelectron spectroscopy (XPS) was utilized to compare the polished/etched CNF sample to the unpolished samples thus to confirm the removal of Ni catalyst prior to the immobilization of ChOx. As expected, no peaks in the Ni region are observed after the mechanical polish and the electrochemical etch. As well, XPS was use to study the electrode before and after the modification with the EDC/sulfo-NHS and ChOx protein. As expected, peaks corresponding to the EDC/sulfo-NHS molecules were observed in the N (1s) and S(2p) binding energy region. On the contrary, the unmodified surface did not show binding energy peaks in these same energy regions. After the addition of the protein, no peak was observed in the S(2p) binding energy region. In contrast, the peak from the N(1s) region increased due to the protein’s presence on the surface of the chemically modified CNF electrodes. Cyclic voltammograms (CV) of CNFs before the modification of the ChOx protein produced quasireversible redox peaks characteristic of Fe(CN)6

-3/-4 redox couple. On the contrary, after the modification with ChOx a decrease in the current is observed due to the presence of the protein at the electrode. .Amperometric response of the ChOx/CNF electrode have been done at a fixed potential of 0.6V vs Ag/AgCl with different addition of 50uL of a 0.5% Cholesterol in 10%Triton stock solution. An increase in current was observed at each addition. However, further experiments have to be done to have a more detailed characterization of this biosensor toward the detection of cholesterol.

• Detailed characterization of the Cholesterol Detection at the ChOx/CNF electrode

• Determination of detection Limit and sensitivity for cholesterol • Cholesterol detection in the presence of possible interference• Covalent Modification of the ChOx with the glycosylation

technique for stability studies

• Hearth disease

• Hypertension

• Arteriosclerosis

• Coronary Artery Disease

• Cerebral thrombosisCholesterol Molecule

Abnormal levels of cholesterol leads to:

EDC

Sulfo-NHS

10mg EDC20mg s-NHS

1mL 0.1M MESpH=62hr

Step #1

10min

Sonication 0.1M MES

1mg/mLChOx0.1M PB pH=7.5

overnight

Step #2

Polished/Etch

Cholesterol Oxidase

National Aeronautics and Space Administration

www.nasa.gov

1

a.u

.

S 2p region

ChOx modified CNF

CNF

EDC/sulfo-NHS

a.u

.

N 1s region

ChOx modified CNF

CNF

EDC/sulfo-NHS

X-ray photoelectron spectroscopy (XPS) high resolution spectra for the S 2p and N 1s binding energy regions for the following samples: etched carbon nanofiber, EDC/s-NHS modified carbon nanofiber and ChOx modified carbon nanofiber. The XPS analysis was conducted using an Al Kα 350W monochromatic X-ray source with a 45ºtake off-angle. A schematic representation of each modification is shown on the right.

X-ray photoelectron spectroscopy (XPS) high resolution spectra for the Ni 2p to a unpolished CNF electrode followed by mechanical polishing and electrochemical etch in 1M NaOH priot to ChOx immobilization to ensure a surface without Ni catalyst.

Xray Photoelectron Spectroscopy

Amperometric Response of the Cholesterol Oxidase modified CNF to 50 µL addition of 0.5 % Cholesterol 10 % Triton. The applied potential was 0.6 V vs Ag/AgCl

Cyclic voltammetry of carbon nanofiber before (a) and after (b) immobilization of cholesterol oxidase in 2.5 mM Fe(CN) 6

-

3/-4 PB pH=7.5. Scan rate 50 mV/s

Cyclic voltammetry of the CNF electrodes recorded in different concentrations of H2O2 in 0.1 M PB pH=7.5 with a scan rate of 50 mV/s. E=0.5V

Cholesterol Detection at Cholesterol Oxidase Modified CNF Electrode

Experimental Parameters and Results