Project proposal for modification of carbone nanotube (cn ts) with metal nanoparticles for electrochemical immunoassay of alpha fetoprotein

By

Awad Nasser Albalwi

Introduction

An immunosensor

multi-walled carbon nanotubes ( MWCNTs)

Alpha-fetoprotein (AFP):

Some Latest review

Objective:

Methodology

Modification Carbon Nanotube with Carboxylic Acid group :

Carbon Nanotubes modified with Magnetic Nanoparticles

Synthesis and bioconjugation of GoldMag-functionalized CNTs

Principle the magneto-controlled electrochemical immunoassay

Caractrization of bio- GMCNTs

An immunosensor is a kind of biosensor that provides concentration-dependent signals by using antibodies (Ab) or antigens (Ag) as the specific sensing element .

Recently, electrochemical immunosensors have incited the interest of scholars because of their sensitivity, highly selectivity, convenience and inexpensiveness, and they have been successfully applied in environmental analysis , the food industry , and clinical chemistry .

-is a major plasma protein produced by the yolk sac and the liver

-The AFP expression is often associated with diseases of

hepatocellular, testicular nonseminomatous origin, and occasionally

of other entodermal origin and has been widely accepted as a tumor

marker for monitoring the therapeutic effectiveness of hepatocellular

cancer and nonseminomatous testicular cancer (Tomasi, 1977).[41]

Most studies report elevated AFP concentrations in approximately 70% of patients with hepatocelllularcarcinoma. Elevated AFP concentrations are found in 50% to 70% of patients with nonseminomatoustesticular tumors.

Normal AFP levels for men for for non-pregnant women are less than 300 nanograms per milliliter.

Greater-than-normal levels of AFP in men and non-pregnant women may indicate:cancer in testes, ovaries, biliary (liver secretion) tract, stomach, or pancreas

cirrhosis of the liver liver cancer malignant teratoma recovery from hepatitis

covalent conjugation of the enzyme to the amino-modified nanoparticle surface. Functionalization of the magnetic nanoparticle surface with amino groups greatly increased the amount and activity of the immobilized enzyme compared with immobilization procedures involving physical adsorption.

In this research proposal , we propose to design a new Gold- Mag-modified CNTs (SWCNTs, DWCNTs and MWCNT) as biosensor for electrochemical detection of Alpha-fetoprotein (AFP) in a flow system (human-blood).

The aim of this work is to explore a new electrochemical immunoassay method using (Gold- Mag-modified CNTs) for the determination of cancer marker in human serum . Hopefully, the methodology can be applied for the determination of real samples with high sensitivity and feasibility in clinical screening of cancer markers and diagnostics.

Scheme 1. Schematic representation of the process of the bio-GMGPs

The multi-wall CNTs will be pretreated according to the procedure with minor modifications According to the method used by (Tang et al 2009, Yu et al 1998, [57,58]. The multi-wall carbon nanotubes (95%) will be oxidized by refluxing in concentrated HNO3 and H2SO4 (volume ratio of 1:3) for 12 h. The resultant mixture then will be diluted with pure water to about three times of the original volume and stirred for 24 h. The mixture will be subjected to high-speed centrifugal sedimentation.

After modifying CNTs with Carboxylic group, Magnetic CNTS can be synthesized according to the literatures with some modification .

1- Amount of CNTs can be initially dispersed into distilled water, and then sonicated for certin time at room temperature .

2- Following that, amount ( mg) of NHS (N -ydroxysulfosuccinimide) and amount (mg) of EDC (N-(3-dimethylaminopropyl)-N -ethylcarbodiimidhydrochlorid) will be added into the mixture, and incubated for certain time at 4 ℃ to activate the –COOH groups on the CNTS.

3-Afterwards, amount ( mg) of amino functionalized magnetic nanoparticles (fluidMAG-Amine) will be added into the mixture, and further stirred for 12 h at 4 ℃. The unconjugated magnetic nanoparticles were removed by filtration for 10 times with the distilled water. The obtained CNTs then will be dried in a vacuum oven at 60 ◦C.

◦ 1- Magnetic CNTS will be dispersed into amount ( mL) of distilled water, and the mixture will be then sonicated for 30 min at RT.

◦ 2- Following that, amount (mL) of gold colloids (C[Au] ≈ 24 mM) will be added into the mixture, and incubated for 6 h at 4 ◦C with slight stirring to make nanogold particles assemble on the Magnetic CNTS surface. Afterwards, the

GoldMag -functionalized Magnetic CNTS will be separated and purified by using an external magnetic field. The obtained GoldMag -functionalized Magnetic CNTS ( it can be called as GMCNTs) will be then dispersed into amount of distilled water, and used for the conjugation of HRP-anti-AFP

◦ 1-Amount of HRP-anti-AFP will be injected into the GMCNTs suspension, and incubated for 6 h at 4 ◦C with slight stirring.

◦ 2- Afterwards, HRP-anti-AFP-labeled GMCNTs (designed as bio- GMCNTs) will be collected through an external magnet. The obtained pellet will be re-suspended into 2.5 wt% BSA(bovine serum albumin ) for 60 min at RT to eliminate non-specific binding effect and block the remaining active groups.

3- Finally, the bio- GMCNTs will be e suspended into certine amount of pH 6.0 PBS(phosphate-buffer saline (PBS)) containing sodium azide, and then can be stored at 4 ◦C .[61] Noticeably , this method was applied for grapheme oxide , and it can be apply for CNTs as a result of the similarity in properties between the two

Scheme 1. Schematic representation of the process of the bio-GMGPs

Scheme 1. Schematic representation of the process of the bio-GMGPs

To verify the successful synthesis of the bio-GMCNTs,

1-(1)transmission electron microscopy (TEM) to characterize the synthesized GMCNTs. Because of Nanometer-sized particles could be formed on the surface of CNTS.

2- the UV–vis absorption spectroscopy of variously functionalized CNTs ( such as (a) CNTs, (b) MCNTS, (c) GMCNTs and (d) bio-GMCNTS Especially, when HRP-anti-AFP molecules are assembled on the GMCNTs. On the basis of the results of these methods , we might make a conclusion that the bio-GMCN Ts could be synthesized by using this new developed method.

According to the method which explained by Zhang et al 2011 and others, we will use AFP as an example to investigate the feasibility of the electrochemical immunoassay. In the experiment, we will investigated the cyclic voltammograms of the bio- GMCNTs-modified electrode in pH 6.0 PBS-[Fe(CN)6]4−/3− solution at the absence and presence of H2O2.

[61]

1- electrochemical immunoassay was carried Electrochemical Analyzer instruments , with a conventional three-electrode system comprising a gold disk working electrode (Au, 3 mm in diameter), a platinum wire auxiliary electrode and an Ag/AgCl reference electrode.

2-The microfluidic device consisted of a six-way valve

equipped with a 1-mL syringe pump and connected through a teflontubing with the flow cell.

3-The gold disk electrode was installed at the bottom of the detection cell, and an external permanent BaFe12O19 magnet with pot shape (10 mm in diameter and 5 mm in depth,

410–430 mT) was set under the gold disk electrode. All the solution was flowed through the detection cell at 100 L min−1.

(i) 100 µ L of the bio-GMGPs (C[GMGP] ≈ 1.5 mg mL−1) was flowed through the detection cell, and collected on the gold

disk electrode with an external magnet; (ii) 100 µ L of AFP standards /or serum samples with various

concentrations was added into the detection cell, and incubated for 20 min at RT without the external magnet; and

(iii) 100 µL of 0.1 M PBS (pH 6.0) containing 5 mM Fe(CN)6]4−/3−

and 1.5 mM H2O2 was injected into the detection cell with an external magnet, and the differential pulse voltammetry (DPV) from 400 to 0 (vs Ag/AgCl) with a pulse

With the aid of an external magnet, the bio-GMGPs will be immobilized on the working electrode.

The carried HRP-anti-AFP molecules on the bio-GMGPs catalyze the

reduction of H2O2 relative to H2O2 - [Fe(CN)6]4−/3− system.

3-Upon addition of the AFP in the detection cell, the analyte reacts

with anti-AFP on the bio-GMGPs.

4-The formed antigen-antibody immunocomplex is coated on the surface of the immunosensing probe.

5- Since AFP and anti-AFP are biomacromolecules with weak conductivity, the formed immunocomplex can act as an inert layer and partly shield the active center of the HRP enzyme, which weaken the catalytic efficiency of the HRP toward the reduction of H2O2 in the solution.

As a result, the current response is decreased. The decrease in the current depends on the concentration of AFP in the sample. The assay principle can be summarized as follows:

Immunoreaction

HRP-anti-AFP +AFP→ HRP-anti-AFP-AFP (1)

Electrochemical measurement

H2O2 +HRP(red)→ H2O +HRP(ox) (2)

HRP(ox) +2[Fe(CN)6]3−→ HRP(red) +2[Fe(CN)6]4− (3)

[Fe(CN)6]4−→ [Fe(CN)]3− + e− (4)

Thank you for your attention