Affinity cantilevers and EIS based biosensors for Cardiac

Diagnostics
Nitin Kale, Ashwani Mehta, Manoj Joshi, Soumyo Mukherji, Rakesh Lal, R.Pinto,
*
P.R.Apte, V.Ramgopal Rao
Centre for Nanoelectronics, Department of Electrical Engineering, IIT Bombay,
Powai, Mumbai-400076
* Email: rrao@ee.iitb.ac.in, Phone: 91-22-25767456

ABSTRACT

Myocardial Infarction (MI) is a serious degenerative disease affecting a large

population worldwide. A reliable method to detect MI requires detection of biochemical
markers such as CKMB, myoglobin and troponin. In this paper we report the affinity
cantilever and the EIS capacitor based sensors to detect myoglobin and CKMB. We
demonstrate cantilever device structures, surface modification techniques and antibody
immobilization. We also report our efforts to detect CKMB using an EIS capacitor. The
device currently works also as a reliable sensor to detect glucose and urea.

1. INTRODUCTION

Acute Myocardial Infarction (AMI), commonly known as the heart attack, is

becoming a major cause of death all over the world. The term Myocardial Infarction is
derived from myocardium (heart muscle) and infarction (tissue death). The phrase "heart
attack" is used to refer to heart problems other than AMI, such as unstable angina pectoris
[1]. The underlying mechanism of a heart attack is the death of heart muscle cells due to lack
of oxygen. The heart muscle cells require oxygen for the metabolic processes. Oxygen is
supplied to the heart by the coronary arteries. If sufficient oxygen is not supplied to the heart
muscle cells, the cells die due to a process called infarction.

World health organization (WHO) criteria have classically been used to diagnose
AMI [7]. A patient is diagnosed with AMI if two (probable) or three (definite) of the
following criteria are satisfied: (i) clinical history of ischaemic type chest pain lasting for
more than 20 minutes (ii) changes in serial ECG tracings (iii) rise and fall of serum cardiac
biochemical markers such as creatine kinase, troponin I and lactate dehydrogenase isozymes
specific for the heart.
 Biochemical markers are macromolecules, which are not present in the blood sample
of a normal person. Fig. 1 shows the various cardiac markers released in the blood after the
onset of AMI. Each of these markers attends its peak at a different time after the onset of
AMI. Considering this aspect, Troponin I, Myoglobin, Creatine Kinase and its isoforms are
commonly quantified in the blood for diagnosis of AMI.

Fig.1 Plot of the appearance of cardiac markers in the blood versus time after
onset of the Acute Myocardial Infarction symptoms [7].

Myoglobin is a specific cardiac dysfunction marker. It is the first marker to be released in the
blood after the onset of AMI. It can be detected within 4 to 30 hours after the onset of AMI.
Creatine Kinase (CK) is an enzyme. Its serum activity exceeds the normal range within 4 to 8
hours after the onset of the AMI, and declines to a normal value after 2 to 3 days. To make
CK more specific to cardiac muscles, three isoforms of Creatine Kinase (MM, BB and MB)
have been identified as biochemical markers of cardiac dysfunction. Only the MB isoenzyme
is specific to cardiac muscles [13]. Fig.1 also shows that troponin is an early marker of AMI.
It is released at about the same time as CK-MB (6 to 9 hours); it is also a late marker as it
remains elevated for over a week after the infarct [14].

2. DETECTION USING AFFINITY CANTILEVERS

Microfabricated cantilever based sensors are widely used for chemical and biochemical
sensing. These cantilevers transduce changes in temperature, mass or surface stress into a
(nano) mechanical response. Antibody immobilization followed by specific antigen binding
or DNA hybridization induces surface stresses in the cantilever due to van der Waals,
electrostatic or steric interactions. These surface stresses bend the cantilever. Bending of the
cantilever can be detected optically [5]. A piezoresistor strain gauge embedded in the
cantilever experiences a change in strain due to the bending and responds with a change in
resistance (electrical detection) [6]. The basic steps involved in this sensor system are (i)
fabrication of a suitable cantilever stack (ii) cantilever surface modification to make one
surface amenable to antibody immobilization (iii) antibody immobilization (iv) interface the
device with AFM (optical detection) or electronics (electrical detection) (v) analyte
introduction followed by detection.

(i) Materials for device fabrication: We have chosen silicon wafer as the substrate material.
The materials for fabricating the device on silicon substrate include silicon dioxide, silicon
nitride, polycrystalline silicon, SU-8 and Chrome-gold. While silicon dioxide is thermally
grown, the chrome-gold film is either sputtered or evaporated.
For depositing silicon nitride and polycrystalline silicon, we have designed and integrated
indigenously a custom made Hotwire CVD cluster tool. Figure 2 shows the tool that has 3
reactor chambers to deposit different materials, the chamber in the centre is the load-lock
cum transfer chamber. We have designed the tool such that substrates can be moved from
one reactor to the other without breaking vacuum. In the hotwire CVD technique, process
gases such as silane, ammonia etc, are dissociated by a tungsten filament, in a vacuum
chamber at roughly 10-7 mbar. The dissociated species are then deposited on the substrate.

Fig. 2: Hotwire CVD Cluster tool Fig.3: Hotwire CVD Silicon nitride beam
(ii) Cantilever structures: Figure 3 shows a silicon nitride beam structure. The silicon nitride
film that forms the structural material of the beam was deposited using the hotwire CVD
process. Figure 4(a) shows a cantilever structure with silicon nitride as its structural material
and Fig. 4(b) shows a stack of gold/nitride cantilever. The gold layer can be used as a
piezoresistive layer in the piezoresistive scheme of detecting surface stress. For a typical
MEMS application the nitride is ultra thin, its thickness being only about 200 nm.

(a) (b)
Fig. 4(a) 200 nm Hotwire CVD silicon nitride cantilevers (b) Gold/nitride cantilevers with gold as piezoresistive
layer
(ii) Immobilization: To immobilize antibodies on the cantilever surface, the surface should be
suitably modified. We have developed a novel surface modification technique that employs
pyrolytic dissociation of ammonia in the hotwire CVD apparatus [7]. The amino groups so
dissociated, are attached to the SU-8 or nitride cantilevers. HIgG antibodies are then
immobilized on the cantilever surface. Finally to confirm immobilization, anti-HIgG proteins
are incubated on the cantilever. The device is then observed under a fluorescent microscope.
Fluorescence seen in the figure is an indicator of attachment of FITC tagged anti-HIgG to
HIgG antibodies on both nitride and SU-8 cantilevers.
 (a) (b)
Fig.5: (a) Silicon nitride cantilever treated with pyrolytic disassociation of ammonia, followed by antibody
immobilization as observed under fluorescence microscope (b) ) SU-8 cantilever treated with pyrolytic
disassociation of ammonia, followed by antibody immobilization as observed under fluorescence microscope

Antibody immobilization via the hotwire CVD technique is being extended to immobilize the
myoglobin antibody on microcantilevers, so as to detect myoglobin.

3. EIS CAPACITOR BASED SENSORS

The Electrolyte Insulator Semiconductor (EIS) Capacitor sensor works on the principle of pH
sensing. In this sensor we measure the catalytic activity (due to control CK-MB) or change in
pH, as the reaction progresses in time. In other words the change in pH is measured as shift
in flat band voltage of the EIS capacitor device. The flat band voltage shift is plotted with
respect to time, and the slope of this plot is indicative of quantity of CK-MB. The established
assay for CK-MB involves a sequence of three reactions catalyzed by 3 enzymes. The first
reaction is hydrolysis of creatine phosphate in the presence of enzyme CK-MB. The ATP
generated in the first reaction is reacted further with glucose under the catalytic action of
hexokinase to give glucose-6-phosphate. In the last reaction glucose-6-phosphate is oxidized
to gluconic acid in the presence of NADP by G-6-PDH enzyme. The reaction liberates
protons into the medium which will lower the pH of the solution. The EIS capacitor sensor
can sense this change in pH by showing shift in flat band voltage. The pH change for the CK-
MB assay system was determined by glass electrode and was found to be 0.01 pH units. The
reactions involved are:
Creatine phosphate + ADP → Creatine + ATP
Glucose + ATP → Glucose-6-phosphate + ADP
Glucose-6-phosphate + NADP → Phosphogluconic acid + NADPH + H +

The sensor was used to sense the CK-MB using the chemicals supplied with the CK-MB
detection kit. Initially the reagent is put on the device and the measurements are taken, after
some time the CK-MB is added and the measurements are taken for some more time. From
the data of this experiment, plot of 'shift in flat band voltage' versus time is obtained. The
offset in slope of curve before and after addition of CK-MB is indicative of the presence of
enzyme CK-MB in the solution.

(a) (b)
Fig. 6(a): Shift in flat band voltage vs time for CKMB (b) Shift in flat band band voltage vs time for glucose

The results in this experiment are shown in Fig 6(a). The result seems to detect the CK-MB,
but there are issues with reproducibility. One reason for this problem could be low reaction
rate constant in third reaction; the other reason could be the presence of buffers present in the
reagent for optimum activity of CK-MB. This divides the further experimentation strategy in
two prongs namely a) to address the reproducibility in experiment set up of point three b) to
bypass the third reaction. To bypass the third reaction calls for the detection of CK-MB using
principle of glucose sensor. First the glucose present in the reagent (containing all the
chemicals present in the following sequence and supplied with the Randox kit) is detected
using EIS capacitor glucose sensor (made possible by allowing glucose to react with glucose
oxidase and hence detecting the resulting pH change). Next, the reagent is allowed to react
with CK-MB in a separate vial for a specific duration of time, this decreases the amount of
glucose present in the reagent (reactions one and two of the reaction sequence), this
decreased amount of glucose is then detected using EIS capacitor sensor. The offset of slope
between the above said two experiment runs should indicate the presence of CKMB.

Creatine phosphate + ADP → Creatine + ATP

Glucose + ATP → Glucose-6-phosphate + ADP
Glucose-6-phosphate + NADP → Phosphogluconic acid + NADPH + H +

In the first phase of the above said experiment an attempt is made to detect the presence of
glucose in plain distilled water with KCL as the support electrolyte. The results of this
experiment are depicted in Fig. 6(b).

The plot of shift in flat-band voltage vs glucose concentration is shown in Fig. 7(a). It is clear
that an increase in the flat band voltage shift is seen, as the concentration of glucose is
increased. However beyond a certain value of glucose concentration, the shift in flat band
voltage saturates. The region of the plot in Fig. 7(a), which is linear, is expanded and
truncated and is shown in Fig. 7(b).

(a) (b)
Fig 7(a) Flat band voltage shift vs glucose concentration (b) same plot showing linear region only

Thus a working glucose sensor is demonstrated. This technique has been extended to also
detect urea. Further work is on the way to reproducibly detect CKMB using this device.
 4. CONCLUSION

Occurrence of AMI can be detected and monitored by detecting biochemical markers such as
myoglobin, CKMB and troponin. Affinity cantilevers were designed and fabricated to detect
myoglobin. Antibody immobilization on silicon nitride and SU-8 surface were demonstrated
using HIgG antibodies and FITC tagged anti-HIgG proteins. Surface modification of
cantilever structures was accomplished through a novel dry technique. Amino groups were
obtained via pyrolytic dissociation of ammonia in a hotwire CVD chamber, which was
indigenously developed. Hotwire CVD silicon nitride was used to fabricate beams,
cantilevers and also strain gauges using gold as the piezoresistive layer. EIS capacitor based
sensors were developed to detect glucose and urea; and this technique is being extended to
reproducibly detect CKMB.

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