Detection of single viruses

Viruses are among the most important causes of human disease.

Detection: Established methods for viral analysis include plaque assays, immunological
assays, transmission electron microscopy, and PCR-based testing of viral nucleic acids.
These methods have not achieved rapid detection at a single virus level and often require a
relatively high level of sample manipulation that is inconvenient for infectious materials.
The ability to detect rapidly, directly, and selectively individual virus particles has the
potential to significantly impact health care.
A two-step procedure was used to covalently link antibody receptors to the surfaces of the
silicon nanowire devices.
First, the devices were reacted with a 1% ethanol solution of 3-(trimethoxysilyl)propyl
aldehyde for 30 min, washed with ethanol, and heated at 120°C for 15 min.
Second, monoclonal antibodies (mAb) receptors, anti-hemagglutinin for influenza A and anti-
adenovirus group III were coupled to the aldehyde-terminated nanowire surfaces by reaction
of 10–100 µg/ml antibody in a pH 8, 10-mM phosphate buffer solution containing 4 mM
sodium cyanoborohydride. (anti-adenovirus group III antibodies, which should have no
specificity against influenza A).

Dr. S. Paria, NIT RKL Nanotechnology 1

 Nanowire-based detection of single viruses

Concept of nanowire-based detection

of single viruses. Schematic
illustration showing two nanowire
devices, 1 and 2, in which the
nanowires are modified with different
antibody receptors. Specific binding
of a single virus to the receptors on
nanowire 2 produces a conductance
change. Right: Characteristics of the
surface charge of the virus only in
nanowire 2. When the virus unbinds
from the surface the conductance
returns to the baseline value

PNAS, 101, 14017-14022, 2004

Dr. S. Paria, NIT RKL Nanotechnology 2
 Cholesterol detection
Normal human blood serum contains less than 200 mg/dl cholesterol, of which two third is
esterified with fatty acids and one third is present as sterol
Mainly enzymatic procedures, using colored substances are employed in clinical diagnosis.
This method usually involves complex procedures for precipitation of lipoproteins
fractions and suffers from low specificity, instability of reagents and high cost. So, it is
desired to develop techniques for cost-effective, convenient, rapid and sensitive estimation
of cholesterol levels in blood.
Electrochemical biosensors using immobilized enzymes on electrodes have become popular
for detection of biological substances. The most critical step in this method is the
immobilization of enzyme onto the electrode surface.
Immobilization procedures such as direct physical adsorption and entrapment in
polymeric films.
Two enzymes are most important: Cholesterol oxidase (COX) and cholesterol esterase
(CE).
These together can be used to monitor both native and esterified cholesterol levels. COX
catalyzes the oxidation of cholesterol, while CE catalyzes the hydrolysis of esterase
esterified cholesterol, which is important for determination of total cholesterol.

Dr. S. Paria, NIT RKL Nanotechnology 3

 Electrode preparation:
The sensor consists of two carbon paste
electrodes. One electrode was used as
working electrode while the other was used
as reference electrode. The function of the
silver leads was to improve electric
conductivity of the electrodes. The reaction
area, where the carbon paste film was
modified by carbon nanotubes and
immobilized enzymes, was defined by the
insulating film coated on the carbon paste
film. The size of the reaction area was
2mm×2mm while the sensor strip was
35mm×10 mm.
Electrode modification and enzyme immobilization
After dropping .2 µl the carboxyl modified multi-walled carbon nanotubes solution (5 mg/ml)
onto the reaction area of the electrode, 1 µl CMC solution (20 mg/ml) was added. It was dried in
hot air at 50 ◦C for 10 min to form a hydrophilic polymer layer. Subsequently, 70 unit cholesterol
oxidase, 1.8 mg peroxidase, 8 mg potassium ferrocyanide and 6 mg trehalose were dissolved in
0.125 ml phosphate buffer of pH 7. Then 2 µl solution was added on the hydrophilic polymer
layer to form a cholesterol oxidase layer. Finally, 200 unit cholesterol esterase, 3 mg trehalose, 1
µl TritonX-100 were dissolved in 0.125 ml phosphate buffer (pH 7). And 2 µl of the solution was
added to form a cholesterol esterase layer.
Dr. S. Paria, NIT RKL Nanotechnology 4
Optimum conditions:
Optimization of electron mediator concentration
The concentration of potassium ferrocyanide, which plays the role of redox mediator has a
certain effect on response current. Proper potassium ferrocyanide increases the response
current while too much potassium ferrocyanide inhibits the reaction.
Optimization of buffer pH
Enzyme has an optimal pH for its activity, but it will alter after immobilization. The optimal
pH was determined by measuring the maximal response current on different pH conditions.
Experimental result shows that pH 7.0–9.0 is optimum to obtain the maximal responses. At
the same time, normal blood is around pH 7.
Optimization of working potential
The sensitivity evidently increased from 100 to 300mV, which is due to the increase of
electron transfer force.
Operating Temperature
The experimental results show that the response current was fairly stable, especially in the
temperature range of 20–30 ◦C.

Dr. S. Paria, NIT RKL Nanotechnology 5

Analysis scheme:
(a) Hydrogen peroxide determination, as result of cholesterol oxidase reaction (1)
(b) Cholesterol esterase reaction (2)
CE
Cholesterol + O2 → 4-cholesten-3-one + H2O2 (1)
Cholesterol ester + H2O COX
→ cholesterol + fattyacids (2)

Hydrogen peroxide is detected by the current response at the enzyme electrode:

H2O2→ O2 +2H+ +2e− (3)
peroxidage
H2O2 + 2[Fe(CN)6]4− + 2H+   → 2[Fe(CN)6]3− + 2H2O (4)
3−
[Fe(CN)6] + e → [Fe(CN)6] 4− (working electrode) (5)
4− 3−
[Fe(CN)6] → [Fe(CN)6] + e (counter electrode) (6)

Dr. S. Paria, NIT RKL Nanotechnology 6

 Using both the carbon nanotube
modified and unmodified sensors, the
responses to cholesterol were
determined over the range of 100–400
mg/dl. An almost linear relationship
between the cholesterol concentration
and the response current of the carbon
nanotube modified electrodes was
observed, while a curve for unmodified
electrodes was observed.
The repeatability of the biosensors is
practically acceptable.

The comparison of the cholesterol

determinations of the carbon nanotube
modified (solid line) and unmodified
Biosensors Bioelectronics 20, 2140-2144, 2005 (dash line).
Dr. S. Paria, NIT RKL Nanotechnology 7
 FETs for Glucose Detection
Similar to other glucose sensors, electrochemical glucose detection is based on
enzymatic glucose oxidation and subsequent hydrogen peroxide detection on the carbon
nanotube electrodes.
The redox enzyme glucose oxidase (GOx) that
catalyses the oxidation of β-D-glucose (C6H12O6) to
Dglucono-1,5-lactone (C6H10O6). Controlled
immobilization of GOx onto the sidewall of a
semiconducting SWNT is found to decrease the
conductance of the tube.
GOx-coated semiconducting SWNTs are found to act as reversible pH sensors and show
an increase in conductance upon adding glucose, suggesting the use as a sensor for
enzymatic activity.
Glucose + O2 GOx
→ Gluconolactone + H2O2.

The quantification of glucose can be achieved via electrocatalytic redox detection of the
enzymatic product H2O2 on the CNT transducer at reduced oxidation or reduction over-
voltage.

Dr. S. Paria, NIT RKL Nanotechnology 8

Real time electronic response of the SWNT
sensor to glucose, the substrate of GOx. The
conductance of a semiconducting SWNT
with immobilized GOx is measured as a
function of time in 5 µL milli-Q water. The
source-drain and liquid-gate voltage were
kept constant at 9.1 mV and -500 mV,
respectively. After 100 sec (red arrow) 2 µ L
milli-Q water was added to the liquid, and
after 200 s (blue arrow) 2 µL 0.1 mM glucose
in milli-Q water was added to the liquid. The
conductance of the GOx-coated SWNT is
observed to increase upon addition of
glucose to the liquid. Inset (a) shows the
same measurement on a second device where
the conductance was a factor of 10 lower.
Inset (b) displays the same measurement on
a semiconducting SWNT without GOx. As
expected, no conductance increase is
observed here.
Nano Lett. 3, 727-720, 2003

Dr. S. Paria, NIT RKL Nanotechnology 9