Direct Electrochemical Detection of Gold Nanoparticle labels for Enhanced Poster Title Immunoassay

Adriano Ambrosi, Arben Merkoi, Anthony J. Killard, Salvador Alegret and Malcolm R. Smyth
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of Chemical Sciences, Anthony J. Ksfsfsf,9, Salvador sfsfsfsft and Msfsfsf Dublin City University, Dublin Ireland Authors: Adriano Ahefsdegi, Arbss Institut Catal de Nanotecnologia, Campus UAB, Bellaterra, Catalonia, Spain Msfsfi, Nanobioelectronics & Biosensors Group, Grup de Sensors i Biosensors, Departament de Qumica, Universitat Autnoma de Barcelona, 08193 Bellaterra, Catalonia, Spain R. sfsfsfsth E-mail: adriano.ambrosi2@mail.dcu.ie
Abstract

of Chemical Sciences, Dublin City University, Dublin 9, and even composite nanoparticles, have been widely Many kinds of nanoparticles, including metal nanoparticles, oxide nanoparticles, semiconductor nanoparticles, Ireland Nanobioelectronics & Biosensors their Institut Catal de Nanotecnologia, Campus UAB, Bellaterra, be used Spain used in electrochemical sensors and biosensors. Due toGroup,unique chemical, physical and electronic properties they canCatalonia, for example to improve the immobilization of Grup de Sensors i Biosensors, Departament de Qumica, Universitat Autnoma de Barcelona, 08193 Bellaterra, Catalonia, Spain biomolecules, enhance the electron transfer, catalyse electrochemical reactions and for labeling biomolecules, achieving in each of those applications, E-mail: azdvzdvno.advzdvzv@mail.dcu.ie signal amplifications and increased sensitivity. The labeling of biomolecules, such as antigen, antibody and DNA with nanoparticles plays an increasingly important role in developing sensitive electrochemical biosensors. Biomolecules labeled with nanoparticles can retain their biofunctionality and interact with their counterparts, and exploiting the electrochemical detection of those nanoparticles, the amount or concentration of analytes can be determined. Dissolution of the nanoparticle labels mostly metal and semiconductor nanoparticles and measuring the dissolved ions with stripping voltammetry represents a general sensitive electroanalytical procedure which, however, involves the use of toxic oxidant solution or dangerous strong acids.
In this work, a sensitive electrochemical immunoassay based on gold nanoparticle (AuNP) labels was developed using a direct electrochemical detection of the particles and therefore without the need of the dissolution step. Streptavidine-modified magnetic beads (MB) were used as supporting material to immobilize a sandwich-type immunocomplex with a biotinylated goat anti-human IgG as a primary antibody, human IgG as antigen and analyte and gold-labelled anti-human IgG as a secondary detection antibody. High sensitive voltammetric stripping analysis was performed to directly quantify the specifically captured gold nanoparticles using a graphite-epoxy composite-magnet electrode (GECE-M), that incorporating inside a tiny magnet, sensibly improved the adsorption of gold on the electrode surface. A comparison with the classical spectrophotometric methods (ELISA) employing HRP-labeled antibodies was also performed.

School

Abstract
A)

Procedure A) Preparation of gold-labelled antihuman IgG using AuNPs (20 nm) and anti-human IgG antibody. B) Assay procedure that consists of the following steps: (I) introduction of streptavidincoated paramagnetic beads (MB); (II) immobilization of the biotinylated antihuman antibody onto MB; (III) first immunoreaction with human IgG; second immunoreaction with either (IVa) gold-labelled anti-human IgG or (IVb) anti-human-HRP. C) Electrochemical analysis of the MBimmunocomplex IVa: adsorption on the electrode surface for 5 min and then DPV catodic stripping analysis (DPCSV) of gold in HCl buffer. D) Spectrophotometric analysis of the MBimmunocomplex IVb: resuspension of the complexes in OPD solution for 2 min; the resulting coloured solution was then separated from the complexes using a magnetic separator and transferred to a cuvette for the absorbance reading at 492 nm.
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+
Au

Anti human IgG

Research Methodologies

B)

Results

IVa
C)

GECE-M

DPV analysis of AuNPs

Conclusion(s)
OPD

II

III
2 min

IVb
D) Results
1 0.9 0.8 0.7 0.6
Current (A)
1e-6 0

UV-Vis analysis

-1e-6 -2e-6 -3e-6 -4e-6 -5e-6 -6e-6 -7e-6 0.0 0.2 0.4 0.6 0.8 1 ug/ml 0.2 ug/ml 0.04 ug/ml 8.0E-03 ug/ml 1.6E-03 ug/ml 3.2E-04 ug/ml 1.3E-05 ug/ml 2.5E-06 ug/ml Blank

Abs

0.5 0.4 0.3 0.2 0.1 0 300 400 500 Wavelength (nm ) 600 700

I (A)
5 4

A
3 0 10 20 30 40 50 60

B
70

Potential (V)

Time (min)

UV-Vis spectrum of Au-NPs (~20 nm, 1.081012 Au-NPs/ mL) prepared by reduction with trisodium citrate. It can be seen the typical absorbance peak of Au-NPs at 520 nm.
1.04

A) Typical DPV curves corresponding to AuNPs analysis for different human IgG concentrations. Deposition at 1.25 V for 120 s; DPV scan from 1.25 to 0 V (vs Ag/AgCl RE); step potential 10 mV; scan rate 33 mV/s; pulse amplitude 50 mV; incubation temperature, 25 C; amount of paramagnetic beads, 150 g. B) Graph of the incubation time optimization. Using magnetic beads the incubation time resulted shortened to 20 min instead of 1hr like in common ELISA test.
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Electrochemical
6

Spectrophotometric

7 6 5 DPV analysis of AuNPs Optical analysis based on HRP

7 6 5 4 3 2 1

0.99
Signal (uA)/D.O.

4 3 2 1

Abs (520 nm)

0.94

0.89

0.84

C
Human (1ug/ml) Goat (1ug/ml) No antigen Human (1ug/ml) Goat (1ug/ml) No antigen

D
1e-6 1e-5 1e-4 1e-3 1e-2 1e-1 1e+0

0.79 0 2 4 6 8 10 12

Anti-human (ug/ml)

Gold aggregation test for anti-human antibody. 7 g/mL resulted the minimum protein concentration necessary to stabilize gold nanoparticles.

Transmission electron micrographs of both MBimmunocomplexes: The pictures above clearly show AuNPs attached to the magnetic beads through the sandwiched immunocomplex IVa. The pictures below of the immunocomplex IVb show a smoother magnetic bead surface.

1e-7

1e+1

Human IgG (g/ml)

C) Non-specific signal study for the electrochemical and the spectrophotometric method, which resulted be of 22% and 6% respectively. D) Calibration curves for the two methods: the electrochemical technique resulted be more sensitive and with a LOD of an order of magnitude lower (Human IgG 0.43 pM) compared to the spectrophotometric one (Human IgG 9 pM).

Conclusions An electrochemical method (DPCSV) to directly detect antibodyantigen interaction based on the use of Au-NPs is developed. Au-NPs are synthesized and then modified with anti-human antibody. The proposed method avoids the use of harmfulprocedures to dissolve AuNPs and can be easily adapted to user friendly and low cost instrumentation. This direct DPV stripping detection method was compared also with the classical spectrophotometric method (ELISA) based on the colorimetric reaction between HRP and the specific substrate OPD, revealing a higher non-specific signal (22% against 6%) but a better sensitivity and a considerably lower limit of detection (0.43 pM against 9 pM of human IgG). Further optimizations in order to lower the non-specific signal are still necessary but the electrochemical method developed based on the detection of AuNP labels represents a valid alternative to the classical spectrophotometric techniques.

Peak current (A)

Abs (492 nm)