CHL036U3E

Biosensors

[Link]

Dharitri Rath
Department of Chemical Engineering
 Recap…

 Different parts of a biosensor

 Biorecognition Element and their properties
 State-of-the art biosensors and engineering challenges
 Types of Transduction events- Optical and
Electrochemical

Strategic consideration for

Biosensor development..

©Dharitri Rath 2
Parts of a biosensor

Transducer

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 Biomedical Devices

1. Microfabrication

2. POC Devices

3. Non-invasive Biosensors

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Point-of-care Diagnostics

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 Introduction

Providing medical diagnostics at or near

patient care
Low resource setting Developed settings

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 POCT Devices

Robust, inexpensive and simple diagnostic tests at

point-of-care settings can greatly improve quality
and efficacy of healthcare available to people
living in developing countries, where the burden of
disease is the highest

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ASSURED Criteria
‘POINT-OF-CARE’ (POC) DIAGNOSTICS

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 How to accomplish this??
Immunoassay-based (ELISA)

State-of-the-art
analytical instrument
[Link]
advantages/

 Multi step processes use a lot of steps and fluid handling

systems
 Use clever engineering to miniaturize diagnostic tests
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 Microfluidics

A system manipulating fluids in channels

having dimensions ~10 to100 micro-meters.

Advantages
 Lower consumption of samples
 Shortens time of experiments
 Parallelization and high
throughput experimentation
The Quake Lab,  Improve the precision of
Stanford experiments
[Link]  Precise fluid control and
[Link]/ analysis of flow and trasnport

©Dharitri Rath 10
 Disadvantages

 Conventional pressure driven microfluidic

devices require significant ancillary equipment

 How to use them as POCT??

©Dharitri Rath 11
 Paper Microfluidics

Fluids are driven by capillary Litmus paper used

pressure generated by to test pH of a solution

porous materials
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Paper-based microfluidic
devices

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 Commercial Success
Home Pregnancy Test

Control
Signal

• Low-cost
• Instrument-free

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Commercial Success
Glucometer

©Dharitri Rath 15
 Common Assay Formats

A: Lateral flow assays , B: Micro-paper analytical devices (μPADs)

C: Paper ELISAs (p-ELISA), D: 2-dimensional paper network devices (2DPN)
©Dharitri Rath 16
J. Ind. Inst. Sc., 98 (2018) 103-136.
Lateral Flow Assays

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Physical Phenomenon

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Gold NP-based Lateral Flow
Immunoassay

Step 1

Step 2

Step 3

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Gold NP-based Lateral Flow
Immunoassay

Au Negative Positive

NP
Detection
Antibody
Control

Analyte Test
Capture
Antibody

Surface

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Fluid Flow Physics

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Darcy’s Law

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 Washburn Equation

Washburn is Valid only for 1D domains

Darcy’s law does not account for imbibition

Both models assume 100% saturation of the pores of the paper

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Richard’s Equation

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 Fluid flow coupled to
Transport of Species

Governing equations
Richards equation Linear velocity

and

Transport of species 
Deff  D

 ( Ci ) r
 .  Deff Ci   u .Ci  Ri  Si  =porosity
t  =Tortuosity

Spatiotemporal variation of concentration of the

reagents could be tracked
©Dharitri Rath
Transport Equations
Rate of accumulation = Input + Generation – Output
– Consumption
Differential volume and mass
V xyz mCi (xyz)
Rate of accumulation in differential mass

Cixyz
Input Output t
 Ci   Ci 
D
 eff ,i vCi x yz
Deff ,i vCi xx yz
x x  x 
  xx

(Ci )
. Deff ,iCi  v.Ci
t
©Dharitri Rath
Commercial Glucose Sensors
and Diabetes Management

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 Commercial Glucose
Sensors
•Biggest biosensor success story!

•Diabetic patients monitor blood glucose at home

•First made by Medisense (early 1990s), now 5 or more

commercial test systems

•Rapid analysis from single drop of blood

•Enzyme-electrochemical device on a slide

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 Diabetes Management
• Insulin secretion by pancreas regulated by blood
glucose, 4.4 to 6.6 mM normal

• In diabetes, regulation breaks down resulting in wide

swings of glucose levels

• Glucose tests tell patient how much insulin to

administer; or what other action to take

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Glucometer

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 Generations
1st generation: The normal product of the reaction diffuses to the
transducer and causes electrical response

2nd generation: Involves specific mediators between reaction and

transducer to generate improved response

3rd generation: Reaction itself causes the response

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 First Generation Glucose
Biosensors
• The first generation glucose biosensors estimated glucose concentration in
the sample based on hydrogen peroxide production by glucose oxidase
utilizing dissolved oxygen.

• Based on this technology, Yellow spring Instrument company, launched the

first commercial glucose biosensor in market in 1975 for the direct
measurement of glucose.

• The usage of the most expensive metal platinum for the fabrication of this
electrode restricted the biosensor to clinical laboratories only.
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 Major Drawbacks of First
Generation Glucose Biosensors
 Amperometric measurement of hydrogen peroxide required a
high operating potential (0.6 V) for high selectivity.

 Restricted solubility of oxygen in biological fluids, which

produced fluctuations in the oxygen tension.

 Deactivation of the enzyme due to the production of hydrogen

peroxide.

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 Second Generation Glucose
Biosensors
The second generation glucose biosensor utilized redox mediator to
transfer electrons from the enzyme to the working electrode surface.

• A variety of redox mediators, such as ferrocene, ferricyanide, quinines,

methylene blue etc were used to improve sensor performance.
• Usage of redox mediator eliminated the need of oxygen for electron
transfer at the electrode surface, thus overcoming the drawback of
limited oxygen pressure observed in the first generation biosensor.
• The lower redox potential of chosen mediators (0-2 V) results in no
interference from other electroactive species such as uric acid, ascorbic
acid.
• Redox mediator enhances the electron transfer between the redox center
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of enzyme and the electrode surface.
 Major Drawbacks of Second
Generation Glucose Biosensors
• High competition between redox mediator and oxygen.

• Interference of other electroactive species lead to false and

inaccurate results.

• Small size and highly diffusive nature of mediators poses

problem of leaching of mediator from intermediate region
between enzyme and electrode surface.

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 Third Generation Glucose
Biosensors
• The third generation glucose biosensors are based on the direct
electron transfer between the active center of enzyme and the
electrode.
• The intrinsic barrier to electron flow is the globular structure of
glucose oxidase with the active site, containing FAD/FADH,
redox cofactor, buried deep inside a cavity of ~ 13 A is a major
hinderance for direct electron transfer.
• Carbon nanotubes immobilized electrode surface provide
suitable orientation for enzyme immobilization and establish
connection between electrode surface and deeply

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Glucometer

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Non-Invasive Methods

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 Non Invasive
The term non-invasive in medicine has two meanings:
A medical procedure is strictly defined as non-invasive when no break in
the skin is created and there is no contact with the mucosa, or skin break,
or internal body cavity beyond a natural or artificial body orifice. For
example, deep palpation and percussion is non-invasive but a rectal
examination is invasive. Similarly, examination of the ear-drum or
inside the nose or a wound dressing change all fall outside the strict
definition of "non-invasive procedure".

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Non-invasive Measurement
• Spectroscopic Measurements on the body to determine one or
more chemical compounds
• Easy to be: Glucose present in slight concentration compared to
other component
• When all components are together- its glucose signal is no long
distinct

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 Samples

• Skin
• Saliva
• Sweat
• Urine

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 Non-invasive Methods
Infrared (IR) light sensor to detect blood glucose levels without puncturing
the skin. These devices typically shine light on the skin and then measure
the IR absorption or transmission wavelength specific to glucose. Other
optical methods use various wavelengths, measurement sites, and
electronic circuitry

Design and Calibration of

calibration these devices
make for has proven to
high unit be a problem
cost

Problems

The calibration period can be quite long

(up to 60 days)
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Interstitial fluid (ISF) glucose
Measurement
Sampling techniques that are
essentially non-invasive, since they
use interstitial fluid (ISF) drawn
through virtually intact skin.
1. One sampling method uses reverse
iontophoresis (electro-osmosis) to
extract the ISF.
2. Another uses a laser to painlessly
create micropores in the dead layer of
skin, through which ISF is collected

MERITS DEMERITS
Small electronic Perspiration, lag
module for time, and other
reading and factors can affect
storing the measurement
electronic data accuracy
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 Iontophoresis involves the
application of a small and defined
electrical current to the skin. This
process causes increased
molecular transport through the
Electro- skin and has found application in Electro-
migration transdermal drug delivery osmosis

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 Electromagnetic Waves
Electromagnetic waves have been
used to sense blood glucose
through the skin noninvasively via
radio-wave impedance
spectroscopy. By varying radio-
wave frequencies, changes in
blood glucose can be determined

Other optical
methods use various
wavelengths,
Calibration can measurement sites,
be problematic and electronic
circuitry.

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Determination of Glucose in
urine
An amperometric glucose biosensor strip
Rapid detection comprises an electrically conductive
measure urine carbon layer, having a first redox
glucose mediator, a reagent strip containing an High sensitivity
concentrations enzyme system for the oxidation of Accuracy
below 3 mg/dl. glucose and a second redox mediator,
and a silver/silver chloride reference
electrode.

A sensing electrode and a reference electrode are arranged so that the

electrically conductive layers of the electrodes are face-to-face and sandwich
the reagent strip between them. Screening for glucose is achieved by
contacting the sensor with a drop of the patient's urine and comparing the
current read-out with a standard calibration curve or by automatically
converting the current flow generated by the test sample to units of glucose
[Link] of urine glucose concentration with the sensor
provides a non-invasive probe of a patient's blood glucose level since a linear
relationship exists between blood glucose concentration and urine glucose
concentration, approximately in the range between 0 to 400 mg/dl.
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Diffuse Reflectance Near-Infrared
Spectrometry for Non-Invasive
Blood Glucose Monitoring
A microcapsule placement unit and
a light detecting non- invasive
reader unit. The microcapsule
placement unit poses the right dose
of glucose- responding
microcapsules in the upper layer of
the skin, painlessly.

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 Fluorescent microspheres
made of polyethylene glycol
A chip-level sensor that uses surface-enhanced Raman spectroscopy to
increase the sensor’s sensitivity. The sensor relies on inelastic scattering
combined with a chemical placed near a roughened metal surface or near gold
or silver nanospheres, resulting in micromolar (1/1000000 molecular weight
per liter), nanomolar, and eventually femtomolar measurements.

Implantable fluorescent microspheres made of polyethylene glycol that have

an assay chemistry specific to glucose that changes the fluorescence work in
conjunction with an external optical biosensor (LED light source) to enable
non-invasive glucose monitoring. (Courtesy Texas A&M) 48
 GlucoWatch Biographer
(First commercially available
electrochemical non-invasive sensor)
Provides:
• Frequent, automatic, and non‐invasive glucose measurements
for diabetic patients

• Glucose readings every 20 minutes for 12 hours

Anal. Bioanal. Chem. 2007, 388, 545‐563

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 How GlucoWatch works?
The current output from this sensor accurately tracked blood glucose
changes and is correlated with the capillary blood glucose values (average
r> 0.93) and a time lag of twenty minutes . The complete electronics: two
sensor potentiostats, an iontophoresis galvanostat, control circuitry,
microprocessor, and LCD display are packaged into a wrist watch format
and are powered by a single AAA battery.

The performance of this device has been evaluated in two large clinical
studies in a controlled clinical environment (n=231), and the home
environment (n=124). Mean difference between biographer and finger-
stick measurements was −0.01 and 0.26 mM for the clinical and home
environments, respectively, while the CV% is around 10%.

Biosens. Bioelectron. 2001, 16, 621‐629. 50

The Gluco-Watch biographer provides frequent measurements of glucose
over a 12-hour period with good clinical accuracy. It can detect trends and
track patterns in glucose levels in diabetic subjects with diverse
demographic characteristics. The GlucoWatch biographer should facilitate
improved diabetes management by the diabetic patient and caregiver.

Time profiles over 12 hours of measurement show close tracking of the biographer results with
fingerstick blood glucose measurements
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[Link]/reviews/evaluation_of_the_glu
 Problems with the
GlucoWatch
• GlucoWatch is not a replacement for standard glucose monitoring and
is never shown to conclusively improve clinical outcomes for users
• After a full two-hour “warm up” period, the GlucoWatch requires daily
calibration with standard test strips and is designed to run over 12-
hour durations.
• The accuracy of the device is questionable.
• The reproducibility of measurements are found to decrease due to
environmental changes(changes in temperature, jostling and
perspiration were all shown to dramatically influence measured blood
glucose concentrations).
• In comparison to standard test strips, the GlucoWatch is significantly
more expensive.
• According to one cost breakdown, the GlucoWatch along with all its
replacable components (battery, adhesive gel electrode pads, etc.) cost
about three times what it would cost to take 8 finger-prick
measurements per day.
• GlucoWatch was uncomfortable for many.
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 A Glucose sensing contact lens: A new
approach to non-invasive continuous
physiological glucose monitoring: feasibility
of physiological glucose sensing in tear
A proto‐type glucose sensing contact lens based on embedded boronic acid
containing fluorophores that responds well to glucose concentrations in the
tear range, 50 – 500 μM glucose.

Daily use
disposable
contact
lenses

Contact lens mount and quartz holder (left). The experimental geometry
used in the sensing study using lens (right)

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 Emission spectra of
DSTBA(4′‐dimethylaminostilbene‐
4‐ boronic acid

Emission spectra of DSTBA in pH 8.0 phosphate buffer‐methanol (2:1) with glucose (A), in lens (B),
λex = 340 nm. Ratiometric response of the probe in buffer and lens with glucose (C)
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 Advantages
• Sensor responses of ≈ 20 % for glucose
• 90 % response time of about 10 minutes
• Does not leach probe
• Has a shelf‐life in excess of the several month daily use

A new technology for the non-invasive continuous monitoring of tear glucose

using a daily use, disposable contact lens, embedded with sugar-sensing
boronic acid containing fluorophores. The continuous monitoring of tear
glucose levels in the range 50 – 500 μM, which track blood glucose levels
that are typically ≈ 5-10 fold higher. The sensing concept with well-
established, previously published, boronic acid probes and the results could
conclude the used probes, with higher pKa values, are almost insensitive
towards glucose within the contact lens, attributed to the low pH and
polarity inside the lens.

A range of probes based on the quinolinium backbone, having considerably

lower pKa values, which enables them to be suitable to sense the
physiological glucose in the acidic pH contact lens have been developed.
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 References
• Non‐invasive biosensors in clinical analysis, George G. Guilbault & Giuseppe Palleschi,
Biosensors & Bioelectronics 10 (1995) 379‐392

• Commercial Biosensors. Applications to Clinical, Bioprocess, and Environmental

Samples, Edited by Graham Ramsay (Wolpert Polymers, Inc). Wiley‐Interscience: New
York. 1998. v + 304 pp. $69.95. ISBN 0‐471‐58505‐X.

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 Summary

• Concentrations of analytes are low in saliva,

sweat ,tears and urine.

• Difficulties in standardization.

• Commercialization

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Biosensors for disease management:
Cancer
 Cancer
 Cancer is a leading cause of death worldwide, accounting for nearly 10
million deaths in 2020, or nearly one in six deaths.
 The most common cancers are breast, lung, colon and rectum and
prostate cancers.
 Cancer is a abnormal and uncontrolled cell growth due to an
accumulation of specific genetic and epigenetic defects, both
environmental and hereditary in origin.

The process of induction of cancer

condition is known as carcinogenesis.
After carcinogenesis, cancer cells
start to produce some specific
molecules, called biomarkers.
 Conventional methods

 Conventional methods for cancer diagnosis include

ultrasound, nuclear magnetic resonance and, biopsy

 Require expensive equipment and highly qualified

personnel

 Biopsy, is an invasive technique that represents a risk for

the patient.
 Are inefficient in the early diagnosis of cancer since they
depend on tumor phenotypic properties.
 Additionally, these techniques have a limit of detection,
thus being incapable of detecting cancer at early stages of
the disease.
 Conventional methods

For protein biomarkers, the most common include western blotting,

ELISA and mass spectrometry; for nucleic acids qRT-PCR,
microarray, and next generation sequencing; and for cell
populations flow cytometry and immunohistochemistry. Despite
these techniques are in usage, they still have limitations like
sophisticated analysis process, time-consuming operations and low
sensitivity.

Biomarkers in body fluids represent a convenient,

noninvasive and cheaper method for cancer diagnosis. Body
fluids including serum or plasma, urine, saliva or sputum
can be used in order to perform the biomarker detection
 Biomarkers
Biomarkers may be proteins, nucleic acids or any biomolecules.
The detection of cancer biomarkers plays a crucial role in cancer
diagnosis, prognosis, and therapeutics.

Biomolecules
(Biomarkers)

Figure: Cancer cell producing biomarkers

 Table: Commonly used
biomarker for cancer diagnosis
and prognosis

I.E. Tothill / Seminars in Cell & Developmental Biology 20 (2009) 55–62

 Molecular Recognition
Entity (Bioreceptor)
Molecular recognition entity is act as receptor and recognize
specifically the target molecule.

It is generally antibodies, oligonucleotides, enzymes, and synthetic

molecular recognition elements.
 Biosensors in the Diagnosis,
Prognosis and Prediction of Cancer

Electrochemical biosensor. An increase in the electrode resistance owed to the

interaction between a biomarker present in body fluids (antigen, complementary
DNA strand or ligand) and its target molecule (antibody DNA single strand or
receptor) attached to the electrode is measured as a change of current.

Ref: [Link]
 Biosensors in the Diagnosis,
Prognosis and Prediction of Cancer

Optical biosensor. Owed to the interaction between a biomarker (antigen,

complementary DNA strand or ligand) present in body fluids and its target molecule
(antibody DNA single strand or receptor) attached to a florescence element, a
quencher release occurs which translates in higher fluorescence.

Ref: [Link]
 Biosensors in the Diagnosis,
Prognosis and Prediction of Cancer

Mass change biosensor. As a result of a change in mass in the piezoelectric device

owed to the interaction between a biomarker (antigen, complementary DNA strand
or ligand) present in body fluids and its target molecule (antibody DNA single strand
or receptor) attached to the piezoelectric device a decay in frequency is recorded.

Ref: [Link]
Multiplexing capabilities: for cancer diagnosis multi-array
sensors would be beneficial for multi-marker diagnosis.

Sample preparation is usually incorporated as part of the sensor

system such as glucose sensors which contain microfluidics and
membrane separation systems for blood samples handling.

The Biosensors are used in cancer management for diagnosis,

Prognosis and therapeutics.
 POCT approaches for
cancer diagnostics
POC systems are integrated sensing platforms that enable one to
run the multistep analytical procedures in one automated single
unit.

This analytical approach, which goes beyond traditional

diagnostic tests supplies the clinician with a valuable source of
molecular information to detect cancer via novel and efficient
sensing platforms.

Using POC devices, bio-diagnostic data can be obtained in a

rapid, economical, and practical manner without requiring skilled
technicians.
Paper-based POC platforms
These systems have been
eagerly adopted in the POCT
fields due to their innate
advantages such as simplicity,
low cost, ease of fabrication,
independence from
complex/external equipment,
and capability of integration
with other analytical devices.

LFA based POC devices have

been successfully employed for
detecting nucleic acid and
protein targets due to their
unique features including short
detection time, and user
friendly formats.
Paper-based POC platforms

The combination of an LFA platform

with a personal glucose meter (PGM)
can be employed for sensitive and
rapid detection of both a small
molecule marker, 8-Hydroxyguanosine
(8-OHdG) and a large protein marker
(PSA) in separate devices.
Paper-based POC platforms

Simultaneous detection of three

types of microRNAs (miRNA-21,
miRNA-155 and miRNA-210) (Fig. a,
b and c) in standard solution and in
spiked serum samples (Fig. d-e).

DOI: 10.3109/07388551.2014.992387
Paper-based POC platforms

Another highly sensitive molecular diagnostic

platforms relies on the new and upcoming
technology which is known as the clustered
regularly interspaced short palindromic repeats
(CRISPR)-associated nuclease (Cas) system. The
CRISPR-Cas are the adaptive immune system in
prokaryotes that uses RNA-guided nucleases to
detect and destroy foreign/invader nucleic acids.
 Printed electrode based
POC platforms
Screen printing technology is one of the methods most favored for
large-scale production of electrochemical POC devices

It is affordable, quick, and compatible with different types of

materials.

According to IDTechEx research, the total market for printed

organic and flexible electronics will rise upto $74 billion in 2030.
Printed electrode based
POC platforms

Figure: Schematic representation of Printed electrode based POC

 Smartphone-based
platforms
Smartphones represent a remarkable new platform for the
development of next-generation POC diagnostics devices

Provide portable and user-friendly mobile healthcare delivery

and personalized IVD systems.

Cellphone technology has opportunities in the area of business

(billions of user) and research.
 Figure: Schematic illustration of the smart-phone based cancer detection
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