By Dr. Kim Van Vliet, Dr. Megha Agrawal, Dr.

Shyamasri Biswas, Contirbuting Editors

Vacuum Technology
Helps Develop Immunosensors for
Advanced Medical Diagnosis

dvances in vacuum technologies and thin films allow for the

generation of novel biomaterials
with unique functional properties for advanced biotechnology and medical applications. These include the development of
novel immunosensor technologies for use
in new analytical devices for a variety of
medical diagnostics applications, such as
acute myocardial infarction, or detection
of clinically relevant mRNA, DNA and
proteins, for example, cholera antitoxin antibody levels. Immunosensors are
compact analytical devices that detect the
event of formation of antigen-antibody
complexes. A transducer then converts
it to an electrical signal that can be processed, recorded and displayed. Vacuum
technology plays pivotal roles in developing such immunosensors by allowing
controlled deposition of thin film coatings
with molecular recognition probes. A
controlled and tailored coating is essential
for the immunosensor analytical device to
function with high efficiency. Several vacuum deposition techniques have been employed to develop immunosensor devices.
For example, chemical vapor deposition
(CVD) and sputter coating techniques
2

have been used to coat thin films on bioactive surfaces. In this column, we will
present some of the important advances
in immunosensors for medical diagnosis.
High-efficiency immunosensors were
developed by using a CVD deposited
graphene monolayer that was grown on
a copper foil using acetylene as a precursor source. These immunosensors were
demonstrated for the detection of human
cardiac troponin I (1). In other studies,
researchers have fabricated a fiber-optic electroconductive surface that was
modified by vacuum deposition of a thin
layer of indium tin oxide. The resulting
biotinpyrrole monomers were then electropolymerized onto the conductive metal
oxide surface and then exposed to avidin.
Subsequently, avidinbiotin interactions
were used to modify the fiber optics with
biotin-conjugated cholera toxin B subunit
molecules, for the construction of an immunosensor to detect cholera antitoxin
antibodies (2).
In other related work, graphene was
directly vacuum coated onto silk fibers.
The researchers produced a graphene silk
composite (G/S) film sensor for glucose
detection by vacuum filtration of a mixed

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suspension of GO and silk fibers, followed by chemical reduction. By drying

the fibers in a GO suspension, they then
converted the GO sheets on the fiber to
graphene, making the insulating silk fibers electrically conductive, as shown in
Figure 1.
High-vacuum and ultra-high-vacuum
magnetron deposition techniques were
employed for protective layering for biomedical device applications [4]. Conformational thin-film aluminum oxide layers were deposited using DC magnetron
reactive sputtering to allow protection
on non-planar geometries. An ultra-high
vacuum DC magnetron sputtering system
was used for metal and aluminum oxide
deposition. Corrosive protection of the
aluminum oxide films was evaluated using lithographically defined metallic device structures that were overcoated with
the developed material and then exposed
to a corrosive fluid such as Phosphate
Buffered Saline (PBS), which is highly
corrosive to metals and is widely used in
biomedical research to simulate the ionic
concentration and osmolarity close to that
of the human body (5). After placing the
samples with the protective aluminum
June 2015 Vacuum Technology & Coating

Figure 1. Schematic illustration of the fabrication of G/S films. a) GO and silk fiber suspension,
b) GO/S film obtained after vacuum filtration, c) G/S film obtained after reduction, d) Digital
photographs of the top and bottom sides of a GO/S film, e) Digital photographs of the top and
bottom sides of a G/S film. Source: Liang et al. (3).

oxide layer in PBS solution for up to 72

hours at room temperature, there was no
visible damage caused to the protected device structures. These samples were also
tested to show that they would not corrode
in biological solutions during an extended
period of electric current application. The
samples with the aluminum oxide layer
were protected during the first 48 hour
period of the test; whereas unprotected
samples showed an increase in resistance
and broke after 48 hours. In these studies,
it was demonstrated that amorphous aluminum oxide film coated with ultra-high
vacuum process was an effective layer
to protect the sensors from corrosion for
at least 48 hours after exposure to a biological solution such as the PBS solution
used in the study. This shows promise for
protecting biomedical device components
from corrosion that would likely occur
due to the nature of biological samples.
Recently, the development of immunosensors has begun to play an important
role in the detection of foodborne pathogenic bacteria, such as Escherichia coli
O157:H7. E. coli O157:H7 can cause
life threatening complications in humans;
therefore, quick, inexpensive, and reliable
means of detection are urgently needed.
Wang et al developed a novel impedimetric immunosensor for the detection
of E. coli O157:H7 using AuNPs to decorate a rGOP electrode (rGOPE/AuNPs).
Then by immobilizing antibodies on this
surface, as shown in Figure 2, the novel impedimetric immunosensor showed

excellent analytical performance for detecting E. coli O157:H7. This high performance immunosensor was easy to fabricate at low cost and shows great promise
for the development of specific sensors
for routine use. This free-standing paper
electrode has the structural and electrical
properties of rGOP and the large active
surface area of AuNPs for antibody immobilization. Wang et al have demonstrated that this immunosensor was specific to
the detection of E. coli O157:H7 and that
this low cost strategy could be adapted for
routine sensing applications.
Mesoporous silica (MPS) is a promising adsorption material, catalyst carrier,
and separation sensor. Biological applications such as drug delivery systems, transfection devices, and regenerative medicines are expected to be developed based
on MPS in the future. MPS particles have
been developed by Yang et al. using vac-

uum deposition for use in electrochemical

immunosensors based on functionalized
MPS particles that were effective for ultrasensitive detection of low concentrations of antigens, 0.01 10 ng/mL (7).
Protein adsorption onto MPS thin films
was also investigated by enzyme-linked
immunosorbant assay (ELISA). For this,
Protein A was allowed to adhere to MPS
thin films that were put in 24 well plates.
The MPS thin films with Protein A were
then treated with FITC-IgG. ELISA tests
using a human IgG ELISA quantitation
kit were performed to evaluate the application of MPS thin films as biosensors.
ELISA is currently used for analytical
biochemistry assays to detect the presence
of a substance, such as an antigen or antibody in a sample. The results demonstrated that a highly effective antigen-antibody
reaction led to high ELISA sensitivity.
The MPS thin film prepared in this study
has the potential for use in highly sensitive
biosensors that will be useful in biomedical applications, such as, the detection of
tumor markers, or the quantitation of endocrine disruptors (8).
A critical tool for the diagnosis and
management of autoimmune diseases is
the ability to measure autoantibodies in
serum. Yet rapid and convenient methods
for point-of-care (POC) detection have not
been achieved, partly due to heterogenous
samples, as well as the very small faction
of the total that these serum autoantibodies represent. Anti-native (double-stranded, ds) DNA autoantibodies are the hallmark of systemic lupus erythematosus
(SLE). POC testing for IgG anti-dsDNA
antibodies will aid clinical diagnosis.
Reliable POC testing for anti-dsDNA

Figure 2. Illustration for the fabrication process of rGOPE/AuNPs based impedimetric immunosensor. Source: Wang, et al (6).

Vacuum Technology & Coating June 2015 www.vactechmag.com or www.vtcmag.com 

and other autoantibodies associated with

rheumatic diseases would provide more
timely results that could improve diagnosis in primary or urgent emergency care
clinics. Recently, Rubin et al developed an
electrochemical biosensor for quantitation
of anti-dsDNA antibodies that performed
similarly to ELISA-based tests (9). An
8-well manifold was machined in two
parts out of acrylic, with an underlying
membrane and a gasket. The bottom piece
has a stainless-steel hollow rod to which
a vacuum can be applied. During the immunoassay, after the final wash step, the
apparatus was disassembled and a plastic
strip containing 8 independently addressable electrode sets aligned with the wells
in the bottom acrylic. The immunoreactant exposed membrane was aligned
with the electrode and the top piece was
sealed to the bottom piece. Each electrode
set consisted of a central working carbon
graphite electrode surrounded by carbon
graphite counter electrode and silver chloride reference electrode. The electrode
terminals were connected through a cable
to an amperometric reader. Anti-DNA is
an important biomarker for diagnosis of
SLE. The reliability of the assay is dependent on the presence of native, dsDNA,
because IgG binding to denatured ssDNA
occurs in normal individuals. Because native DNA is hydrophilic, it will not bind
to hydrophobic surfaces such as PVDF
membranes or polystyrene microtiter
plates. In this system, Ruben, et al used
a synthetic polypeptide pre-coating agent
that binds to the PVDF solid support
through its hydrophobic tryptophan sidechains and to native DNA by electrostatic
interactions between the polypeptide lysine residues and DNA phosphates (9).
This device had superior performance
based on assay speed, low background,
stability, and good correlation with established methods to measure disease
relevant autoantibodies. This portable biosensor to measure anti-DNA antibodies
in the serum of patients with autoimmune
disease uses the electrochemical detection
of TMB substrate by peroxidase conjugated secondary antibody. For the fluidics
requirement, a vacuum source is needed,
and ample wash solution remains a limitation for this device, but its ability to
produce reliable results in approximately
4

30 minutes demonstrates its promise as a

POC device for measuring anti-DNA and
other autoantibodies in a rheumatology
setting (9).
Advances in vacuum technologies allow for the development of new analytical devices for biomedical or biological
applications. The miniaturization of components has resulted in a trend towards
developing point of care (POC) devices
for biomedical applications. Here we
have highlighted several newly developed
immunosensors for biological applications, such as detecting glucose levels
for diabetes patients, immunosensors for
detecting antigens from the foodbourne
pathogens E. coli O157:H7, or Salmonella typhimurium, the use of MPS thin films
for protein adsorption for rapid ELISA assays, and an immunoassay for diagnosing
autoimmune disorders like systemic lupus
erythematosis (SLE). For biomedical applications, time is of the essence and these
vacuum technologies will provide accurate, rapid test results to provide early detection, which will allow for medical intervention to occur sooner in the course of
disease, which generally results in a better
prognosis for patients. Traditional testing
methods for microbial pathogens require
time, usually 24 48 hours to grow cultures of the microorganism. These new
technologies will allow for rapid testing
and isolation of the source of the pathogen
without the need for culturing the microbe
first. Some of these new device components may be in contact with biological
samples. Aluminum oxide thin films to
protect the components of these devices
will allow for repeated use of the devices
over time without corrosion. These technological advances based on the utilization of vacuum deposition processes will
allow for a rapid expansion of point of
care (POC) devices for biomedical applications in the near future allowing personalized medicine to become a reality.

2. Development of an Electroptode Immunosensor: Indium Tin Oxide-Coated Optical Fiber Tips Conjugated with an Electropolymerized Thin Film with Conjugated
Cholera Toxin B Subunit. Konry, T., Novoa, A., Cosnier, S., and Marks, R.S. Anal.
Chem. 2003: 75(11):2633-2639.
3. 
Fabrication and application of flexible
graphene silk composite film electrodes
decorated with spiky Pt nanospheres. Liang, B., Fang, L., Hu, Y., Yang, G., Zhu, Q.,
and Ye, X. Nanoscale. 2014: 6:4264-4274.
4. Development of Pinhole-Free Amorphous
Aluminum Oxide Protective Layers for
Biomedical Device Applications. Litvinov,
J., Wang, Y-J., George, J., Chinwangso, P.,
Brankovic, S., Willson, R.C., and Litvinov,
D. Surf. Coat Technol. 2013: 224: 101-108.
5. Biology of human sodium glucose transporter. Wright, E.M., Hirayama, B.A.
Physiological Reviews. 2011: 91:733-794.
6. Impedimetric immunosensor based on gold
nanoparticles modified graphene paper for
label-free detection of Escherichia coli
O157:H7. Wang, Y., Ping, J., Ye, Z., Wu,
J., Ying, Y. Biosensors and Bioelectronics.
2013: 49:492-498.
7. 
Multifunctional
mesoporous
silica
nanoparticles as labels for the preparation
of ultrasensitive electrochemical immunosensors. Yang, M., Li, H., Javadi, A., Gong,
S. Biomaterials 2010: 31:3281-3286.
8. Preparation of mesoporous silica thin films
by photocalcination method and their adsorption abilities for various proteins. Kato,
K., Nakamura, H., Yamauchi, Y., Nakanishi, K., Tomita, M. Materials Science and
Engineering C. 2014: 40:42-48.
9. Electochemical biosensor for quantitation
of anti-DNA autoantibodies in human serum. Ruben, R.L., Wall, D., Konstantinov,
K.N. Biosensors and Bioelectronics. 2014:
51:177-183.

References
1. Pt nanoparticles-chemical vapor deposited
graphene composite based immunosensor
for the detection of human cardiac troponin I. Singal, S., Srivastava, A.K., Biradar,
A.M., Mulchandani, A., and Rajesh. Sensors and Actuators B: Chemical. 2014:
205:363370.

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June 2015 Vacuum Technology & Coating