Biosensors

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 Key concepts

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 Detect a virus, and the antibodies

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 Polymerase chain reaction

Nucelotide, nucleic acids (RNA, DNA), protein

Adenine (A), thymine (T), guanine (G), and cytosine (C).

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 Antibody (Ig)
An antibody (Ab), also
known as
an immunoglobulin (Ig)

Immunoglobulin M
(IgM): Found mainly in
blood and lymph fluid, this
is the first antibody the
body makes when it fights
a new infection.
Immunoglobulin G
(IgG): This is the most
common antibody. It's
in blood and other body
fluids, and protects
against bacterial and
viral infections. IgG can
take time to form after
an infection.

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 Structure of the antibody

Fab (fragment, antigen binding) region

Fc (Fragment, crystallizable) region
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 Antibody of different species
Covid-19 protein

(Fab)
(Fc)

Covid antigen

Human COVID antibody (IgM)

Anti human IgM antibody

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 Antibody for the same antigen can be different

Because antigen has multiple binding sites available…

Read more: https://www.youtube.com/watch?v=DYc0rlmz0sA

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 The nature of the binding

➢ B cells produce antibody molecules.

➢ Antibodies bind antigens through weak chemical interactions, and bonding
is essentially non-covalent.
➢ A dynamic equilibrium exists for the binding. For example, the reaction is a
reversible one, and can be expressed as

where K is the equilibrium constant.

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 Aptamer

Aptamers are lab-synthesized short, single-stranded DNA or RNA (ssDNA or ssRNA)

molecules that can selectively bind to a specific target, including proteins, peptides,
carbohydrates, small molecules, toxins, and even live cells.

https://doi.org/10.1016/j.omtn.2018.08.023

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 Antibody vs Aptamer

https://www.basepairbio.com/aptamers-vs-antibodies/
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 ELISA (Enzyme-linked Immunosorbent Assay)

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 Direct ELISA

Ref: https://www.youtube.com/watch?v=ohIDilaG16I&t=1s

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 Indirect ELISA

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 Elements of a biosensor

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 Being small and wearable is the trend

system miniaturization

MEDE 4500Shiming Zhang

 Being small and wearable is the trend

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 Being small and wearable is the trend

Skin-like electrodes, smart bandages… Integrated wearables

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 Biosensors
International Union of Pure and Applied Chemistry (IUPAC) definition:
“A self-contained integrated device which is capable of providing specific quantitative
or semi-quantitative analytical information using a biological recognition element
which is in direct spatial contact with a transducer element.”

Mostly the concentration or activity of chemical species in any type of sample.

• Biosensors harness the powerful molecular

recognition properties of living systems and
engineer these into electronic devices to provide
(easy-to-use) sensing with applications in:
• Clinical diagnostics Pregnancy test
• Biomedical research
• Drug discovery Blood glucose meters
• Environmental monitoring
• Food content, quality and safety
• Security and defence
Biosensors for drug discovery DNA microarrays
1. Daniel Thevenot, Klara Toth, Richard Durst, George Wilson. Electrochemical biosensors: recommended definitions and classification.
Pure and Applied Chemistry, De Gruyter, 1999, 71 (12), pp.2333- 2348.
2. Anthony P. F. Turner, Biosensors: sense and sensibility, Chem. Soc. Rev., 2013, 42, 3184
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 Example: Diagnosis of dengue infection
• The most important arthropod-borne
(arbovirus) viral infection of humans
• A global threat and is endemic/epidemic
in nearly every countries in the tropics:
• ~2.5 billion people worldwide are at risk
of infection.
• > 50 million infections each year
• 500,000 hospitalizations (mainly
children), with fatality rate > 5%

• Dengue Map: https://www.healthmap.org/dengue/en/

• Dengue fever is transmitted by the bite of an Aedes mosquito

infected with a dengue virus. The mosquito becomes infected
when it bites a person with dengue virus in their blood.

• http://www.who.int/news-room/fact-sheets/detail/dengue-and-severe-dengue
• Dengue: a continuing global threat, Nature Reviews microbiology, S7, 2010

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 Example: Diagnosis of dengue infection

• Viral RNA detection - based on reverse transcription polymerase chain reaction

amplification of the viral RNA
• NS1 (a dengue protein) detection – NS1 is produced in large quantities during dengue viral
replication (It can be detected as early as the first day the patient experiences a fever)
• Antibodies (IgM and IgG) detection in blood - This (indirect) method is commonly used to
diagnose dengue in the later stage of the disease, after the viral levels have decreased.
(Antibodies against dengue can be detected five days after the onset of symptoms, and IgG can be detected for
many months and even years after an infection)

**serological methods (also called antibody tests)

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 Example: Diagnosis of COVID-19

RT-PCR CRISPR

ELISA Lateral flow

Immunoassasy

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 Components of a Biosensor
provides selective molecular recognition

Enzyme

Antibody

Microorganis
m

Cell

Generally a biosensor consists of two parts:

– A biological recognition element (e.g. enzyme, antibody) to provide selectivity to
sense the target of interest (referred to as the analyte)
– A supporting element which also acts as a transducer to convert the biochemical
reaction into “signal” that can be read out
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 Classification of biosensors
• Biosensors can be grouped according to their biological elements
or their transduction elements.
Biological elements:
• Enzymes
• Antibodies (e.g. immunoassay)
• DNA (e.g. DNA microarray)
• Micro-organisms
• biological cells, tissue, & organelles
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Transducer elements
•Electrochemical → Current, potential,
impedance
•Optical → florescence, light scattering
•Acoustic/piezeoelectric → Mass
•Calorimetric → temperature
•Magnetic
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 Another common classification of biosensors
• Catalytic biosensors:
– Measure steady-state concentration of a transducer-detectable species formed/lost
due to a biocatalytic reaction
– Monitored quantities: (i) rate of product formation, (ii) disappearance of a reactant,
(iii) inhibition of a reaction
– Biocatalysts used: (i) enzymes (ii) microorganisms, (iii) organelles
– E.g. blood glucose biosensor

• Affinity biosensors:
– Devices in which receptor molecules bind
analyte molecules “irreversibly”, causing a
physicochemical change that is detected by
a transducer
– Receptor molecules: i) antibodies ii) nucleic
acids iii) hormone receptors
– E.g. Immunoassay, DNA microarray

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 Catalytic biosensors:
e.g. Electrochemical biosensors
Analyte Product

• Such sensors operate by reacting with the Enzyme

analytes (assisted by enzyme) and Electrode e-
e-
producing an electrical current or voltage
proportional to the analyte concentration.
Apply electrical Measure current
• As certain chemical species (e.g. enzymes) voltage ( analyte concentration)
are oxidized or reduced (redox reactions)
at inert metal electrodes, electrons are
transferred to or from the electrode.

• When a fixed potential is applied to the

electrochemical cell, and a corresponding current, due
to a reduction or oxidation reaction can be used to
quantify the species involved in the reaction.

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 Affinity biosensors :
Immobilization of biorecognition element
• Biological recognition element (ligand) can be immobilized directly to the sensor
surface, or indirectly via an immobilized capturing molecule.

analyte
analyte

biorecognition
element
biorecognition
element capturing
molecule

• One common example of “indirect” capture: Streptavidin-biotin binding

• It provides an alternative to other ligand binding methods e.g. covalent amine or
thiol coupling, and is particularly valuable for applications in molecular biology .

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 Streptavidin-biotin binding -
A “molecular glue” widely used in biotechnology
Microfluidic flow
“biotinylated” ligand analytes
(contains analytes)

Streptavidin (a protein)

Biotin (vitamin H)

• Biotin is often chemically linked to proteins (molecules) for biochemical assays.

• Its small size means the biological activity of the protein (molecules) will mostly
be unaffected.
• Biotin-binding to streptavidin & avidin is resistant to extremes of heat, pH….
• The strong streptavidin-biotin bond can be used to attach various biomolecules to
one another or onto a solid support.
→ Possible to capture biotinylated molecules in a wide variety of environments
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 Affinity biosensors :
e.g. Piezoelectric biosensors
association

Frequency
change
Equilibrium
dissociation
Time Time Time

• They translate a mass change from a chemical adsorption event to electrical

signal . Example: Quartz Crystal Microbalance (QCM)
• Crystal vibrates at resonant frequency parallel to applied field (Typical: 5 MHz ;
research grade: 100-200MHz) : ν = (k/m)1/2
• A change in quartz mass (due to adsorption) changes the vibration frequency.
• Advantage: high sensitivity-- 10’s of ng/cm2
• Disadvantage: highly sensitive to nonspecific adsorption
http://www.sciencedaily.com/releases/2010/11/101124153101.htm 30
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 Affinity biosensors : e.g. Optical biosensors
Input light Reflected/scattered light

Analytes
luminescence/
Fluorescence

Enzymes,
antibodies
Transmitted light
• Based on the measurement of luminescence, light absorbance, reflectance, or
fluorescence emissions that occur in the ultraviolet (UV), visible, or near-infrared
(NIR) → both in solution assays and in test strips.
• Changes in light intensity correlate with the dynamics (binding) and quantity of the
analytes in the biosensor.
• Light signal may come from:
• Directly the light-analyte interaction → “Label-free” techniques
• the optical labels and probes which are specifically tagged to the analytes for
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identification → “labeled” technique
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 Optical biosensor
• Key components: Analytes
• Light sources: LEDs, lasers, Arc lamps….
• Light delivery: free space or optical fibers
(or in general optical waveguides)
• Light detection: photodiodes, image
sensors (CCDs/CMOSs) , PMTs, eyes. Optical transducer
Light delivery
media (light intensity signals:
(free space or fibers) fluorescence, reflectance,
absorbance…)

Surface Plasmon
Resonance (SPR) biosensor Fiber optic biosensor (fluorescent,
(reflectance signal) reflected, transmitted light signal) DNA microarray
(fluorescent light signal)
• Optical Biosensors for the Detection of Pathogenic Microorganisms, Trend in Biotech., 34, 1, P7-25, 2016
• Sensitive optical biosensors for unlabeled targets: A review, analytica chimica acta 620 (2008) 8–26 32
MEDE 4500 • Optical biosensors in drug discovery, Nature Reviews Drug Discovery volume 1, pages 515–528 (2002)
 Example:
Surface Plasmon Resonance (SPR) biosensor
formerly

https://www.cytivalifesciences.com/en/us/shop/protein-analysis/spr-
label-free-analysis/systems/biacore-t200-p-05644
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 Surface Plasmon Resonance (SPR)

•Plasmons:
–collective oscillations of the “free electron gas”
density, often at optical frequencies.

•Surface Plasmons:
–plasmons confined to surface (interface)
–propagating electron density waves occurring at
the interface between metal and dielectric.

•Surface Plasmon Resonance:

–light () in resonance with surface plasmon
oscillation

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 SPR Principle

https://www.youtube.com/watch?v=sM-VI3alvAI

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 Surface Plasmon Resonance (SPR)
Sample Surface Plasmon
medium Wave

Gold Evanescent Wave

Glass

The reflected light

within this narrow cone
angle gets attenuated.

•When the wave vector (kx) closely matches that of the surface plasmon (ksp) at the
metal-sample interface, reflected light is significantly attenuated.
•Within such angle range, significant energy is transferred from the light to the
surface plasmon → resonance effect.
•The resonance angle depends on the local refractive index (n2)
•n2 depends on the local mass density of the sample medium surface.
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 SPR Biosensor
Working priniciples:
Resonance angle changes upon
ligand-analyte interaction
→local mass density change
→refractive index change
→ resonance angle change
→ Reflected intensity change

Label free sensing technique

• High sensitivity
• Simple light coupling

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 SPR Biosensor

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 • In real applications, we often look at populations of proteins, peptides and
nucleic acids
→ Monitor interactions between protein-protein, protein-peptide and
protein-nucleic acid
→ Multiple measurements are required!
→ So, how should we speed up the entire measurement if we have, e.g. 400
different conditions to look into?

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 Biacore sensor chip
A full interaction profile from every spot

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 Key metrics of a biosensor
1. LINEARITY
Linearity of the sensor should be high for the detection of high
analytes concentration.
Measurement
accuracy 2. SENSITIVITY
Ability of detecting minute quantity (concentration) of the
analytes.
A trade-off very 3. SPECIFICITY
common to many Detected signal should solely come from the analytes (or the
biosensors corresponding reactions/bindings). Chemical interference must
be minimized for obtaining the correct result.

Measurement 4.MEASUREMENT (RESPONSE) TIME

Should be as short as possible.
throughput

Examples:
• SPR sensor •ELISA
→Analysis time: 10s → Analysis time: 60 min
→Limit of detection: 3nM →Limit of detection: 0.1 pM

J.L. Arlett, et. al. NATURE NANOTECHNOLOGY ,6, pp. 203, 2011 41
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 Sensitivity versus time (cost) –
A lesson learnt from COVID-19

• Clinical tests are designed for use with symptomatic people, do not need to be low-cost, and require high
analytic sensitivity to return a definitive clinical diagnosis given a single opportunity to test.
• Transmission of SARS-CoV-2 appears to occur days after exposure, when the viral load peaks
➔ This timing increases the importance of high test-frequency and LOW cost.
➔ test must be used at the beginning of an infection to stop onward spread,
➔ reduces the importance of (very) high sensitivity detection
• Tests used in effective surveillance regimens (e.g. intended to reduce the population prevalence of a
respiratory virus during this COVID-19), need to return results quickly to limit asymptomatic spread
➔ sufficiently inexpensive and easy to execute to allow frequent testing (multiple times per week)
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 The importance of early detection

https://www.mdpi.com/2072-666X/11/3/306/htm

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 Immunoassay
• An analytical method using antibodies as reagents to quantitate specific analytes.
• The analytes being measured may be
• those are naturally present in the body (such as a thyroid hormone),
• those that the body produces but are not typically present (e.g. cancer antigen)
• those that do not naturally occur in the body (such as an abused drug).
• Highly specific “lock and key” system: antibody – antigen reaction
• Highly sensitive: can detect proteins at picomolar to nanomolar (10-12 -10-9 moles/liter).
• Simple, precise, and inexpensive(?)

Clinical applications:
• Detecting potential food allergens
• milk, peanuts, walnuts, almonds and eggs
• Disease outbreaks- tracking the spread of disease
• e.g. Zika, Ebola, HIV, bird flu, SARS, colds, STD etc
• Detections of antigens
• e.g. pregnancy hormones, drug allergen, genetically modified organism (GMO),
mad cow disease
• Detection of antibodies in blood sample for past exposure to disease
• e.g. Lyme Disease, trichinosis, HIV, bird flu
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 Some key (layman) definitions
• An antibody is a protein that is produced by the body in
response to an “invading” (foreign) substance. Antibodies are
produced as part of the body’s immune response to protect
itself, e.g. some immunoassays test for the presence of
antibodies to cancer molecules, i.e if the antibodies are
present→ invading cancer cells are, too.

• An antigen is the substance that the body is trying to “fight

off” (eliminate or reduce) by mounting an immune response.
In a test to measure the concentration of a therapeutic drug,
for example, the drug is the antigen that binds to the
antibody.

• An analyte is anything measured by a laboratory test. In immunoassay testing, the

analyte may be either an antibody, or an antigen.

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 Common Testing Methods
• Two Primary Immunoassay Methods
– Immunochromatography - Lateral Flow Device
• Qualitative → Yes/No Results
• few minutes
• Simple procedure for on-site testing anywhere

– ELISA - Enzyme Linked Immunosorbent Assay

• Qualitative or Quantitative
• 1-2 Hours
• Read with or without strip/plate reader
(in clinical laboratory)

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 Immunoassay - Key components
• Specific Antibodies
– Example: differentiate between GMO proteins
• Pure Antigen (target compound/protein)
– Typical analytes in assay measurement
• Enzyme conjugate
– An identifying marker attached to the antibody, used to signal the antigen’s presence.
Enzymes provide detectable signal via their activity; reaction with a specific substrate
chemical yields a colored, light-emitting, or fluorescent product.
• Immobilization substrate
– A physical structure to adhere antibodies

substrate chemical
One example: enzyme

Antigen

Antibody

Immobilization substrate
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 Generic ELISA protocol
1. Coat solid phase with either antibody or analyte.
2. Block remaining binding sites on the solid phase.
3. Add either analyte or “anti-analyte” antibody to be detected.
4. Wash out excess reagent. This separates bound from free analyte.
5. If reagent in step 4 is an analyte, add a second anti-analyte antibody with
detection molecule attached.
6. Wash out excess reagent.
7. Add substrate chemical. The color change or amount of light emitted is
proportional to the level of target analyte.

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 Common types of ELISA:
Immobilization (capture) methods
• Direct - immobilization of the antigen of interest can be done by direct
adsorption to the assay plate
• Indirect - immobilization of the antigen of interest can be done indirectly via
a capture antibody that has been attached to the plate.

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 Common types of ELISA: Detection methods
• Direct detection:
– It can be performed with antigen that is directly immobilized on the assay
plate or with the capture assay format.
– Direct detection is not widely used in ELISA but is quite common for
immunohistochemical staining of tissues and cells.
Direct capture Indirect capture
Direct detection Direct detection

Also known as “Sandwich” assay: very specific because

the antigen must react with 2 antibodies to be detected
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 Common types of ELISA: Detection methods
• Indirect detection:
– Uses a labeled secondary antibody for detection and is the most popular
format for ELISA. The secondary antibody has specificity for the primary
antibody. **Secondary antibodies are generated by immunizing a host animal with an antibody
from a different species, e.g. anti-mouse antibodies are raised by injecting specific purified mouse
antibody into an animal other than a mouse → Goat, donkey, sheep, chicken and rabbit.

Direct capture Indirect capture

Indirect detection Indirect detection

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 Chromogen and fluorochrome labels
• Labels are conjugated (joined) to antibodies in order to visualize the binding of the
antibody. Detection methods include:
• Fluorescence: Fluorescent labels emit light in the visual range when excited by light of a
specific wavelength.
• Colored precipitate: when combined with the appropriate substrate the enzymatic
labels HRP and AP form a colored precipitate.
• ELISA signals could be interpreted by eye or analyzed by spectrophotometer
(spectrophotometry).
Fluorochrome Excitation Emission
(nm) (nm)
Aminomethylcoumarin 353 440
Horseradish peroxidase Alkaline phosphatase (AMCA)
(HRP) (AP) Fluorescein (FITC) 495 528
AEC (red) Fast red (pink) Fluorescein (DTAF) 495 528
Rhodamine (TRITC) 550 570
DAB (brown) INT (yellow / brown)
Texas Redtm 596 620
NBT (brown / purple) Cy2tm 489 505
New Fuchsin (red) Cy3tm 552 565
Cy3.5tm 581 596
TNBT (purple)
Cy5tm 650 667
Vega red (pink) Cy5.5tm 678 703
Cy7tm 743 767
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 Common types of ELISA: Detection methods

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 ELISA read-out

• ELISA's are run in 96 well plates which

are scanned by automated devices.
• 8 rows and 12 columns. Each well holds
approximately 350 ml of volume.
• 384-well and 1536-well plates have been
developed with the same overall
dimensions as the traditional 96-well
plates → for high throughput screening.
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 ELISA read-out
Absorbance

Absorbance

• The color changes/fluorescence emission, and thus the associated binding activities,
are analyzed by spectrophotometry

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 Lateral flow (immuno)assays (LFA)
• Mostly used for qualitative, semiquantitative and to some extent
quantitative monitoring in resource-poor or non-laboratory
environments.
• Applications include tests on pathogens, drugs, hormones and metabolites
in biomedical, veterinary, feed/food and environmental settings, e.g.
Detecting the hormone, human chorionic gonadotropin (hCG), which is the
biomarker for pregnancy and is found in urine and blood

• Key advantages:
• High sensitivity, specificity, good stability
• Relatively low cost and short timeline for development and approval
• Minimal education required for users and regulators

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 Flow
General configuration

• Key feature of LFA is the movement of a liquid sample containing the analyte of
interest, along a membrane strip of polymeric material (e.g. nitrocellulose, nylon,
polyethersulfone, polyethylene or fused silica) thereby passing various zones where
molecules (mostly antibodies) have been attached that will have specific
interactions with the analyte.
• Typical LFA format consists of a surface layer to carry the sample from the sample
pad via the conjugate release pad along the strip crossing the detection zone (at
least has two “Read-out” lines: test line and control line) up to the absorbent pad.
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 Lateral flow (immuno)assay (LFA)
• The particles are typically colloidal gold, or a colored,
fluorescent, or monodisperse latex microbead.

• The particles are conjugated to the specific biological

component of the assay, either antigen or antibody
depending on the assay format.

Target capture antibody: specific for sample analyte

Control capture antibody: specific for conjugated

particles, regardless the presence of target analyte

Conjugated particles: specific for sample analyte

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 Lateral flow (immuno)assay (LFA)

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 LFA: Negative result
• Analytes (e.g. Antigen) not present in sample, conjugated particles pass
test/target line without being captured, but are caught by control line.

Sample (e.g. whole blood, serum,

urine, saliva, cerebral spinal fluid…)
• Liquid moves by the
capillary force of the strip
material.
• Absorbent pad attached at
the distal side of the strip
helps wick the liquid to the
end of the strip, thus
maintaining the flow.
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 LFA: Positive result
• If antigen is present in sample, it will first bind to conjugated particles.

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 LFA: Positive result
• Conjugated particles is remobilized and subsequently bind to antibodies at test line.
• Note that the conjugated particles will bind to control line whether or not the antigen is
present.

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 Covid-19 LFA

J. Med. Virol. 2020, 92, 1524–14518 63

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 Blood Glucose Sensor

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 Diabetes No Longer A Rich-Country Problem

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 Facts about diabetes

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 The most successful biosensors…
• Glucose biosensors still account for >80% of the current world market for biosensors
– Global glucose monitoring device and diabetes management market was valued at USD
8.35 billion for 2017.
• Cornerstone of diabetes management in the past few decades.
– In 2017, the International Diabetes Federation (IDF) estimated that more than 425 million
people have been diagnosed with the disease.
– > 60% of adults with diabetes checked their blood glucose at least once a day.

Ames Reflectance Meter (1969)

Required 50 microliters of blood
(Current meters use 0.5 or less) 67
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 Insulin
• Insulin is a hormone produced in the
pancreas, and released when stimuli from
ingested protein or glucose in the blood
from digested food are detected.
• The insulin cells (beta cells) of the
pancreatic islets prevents high glucose
concentration by sensing its levels and
secreting insulin accordingly.
• Insulin causes cells in the liver, muscle, and
fat tissue to take up glucose from the blood,
storing it as glycogen in the liver and
muscle.
→ Lower the blood glucose level.

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 Insulin
• It helps the blood glucose regulation which is of paramount importance of
global human metabolism ➔ High blood glucose levels→ diabetes mellitus.

Daly et al. Am J Clin Nutr 67 (6): 1186. (1998) 69

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 Diabetes mellitus (diabetes)
Three main types of diabetes
• Type 1 diabetes
– The pancreas fails to produce insulin, and the
person requires to inject insulin.
– Also called insulin-dependent diabetes mellitus
(IDDM), or juvenile diabetes.

• Type 2 diabetes
– The pancreas does not produce enough insulin
or the body cells do not use insulin properly.
– 90% of people with diabetes have Type 2.

• Gestational diabetes
– Pregnant women, who have never had diabetes
before, have a high blood glucose level during
pregnancy.
– It may precede development of type 2 DM.

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 Target Blood Glucose Ranges
• A fasting plasma glucose level : 70–130 mg/dL
(3.9-7.2 mmol/L)
• After meals: less than 180 mg/dL (10 mmol/L).

• Diabetics need to keep careful control of their

blood glucose to prevent either
– hypoglycemia – low blood glucose levels
– hyperglycemia – too high blood glucose levels.

**Keeping your blood sugar in range will

lower your risk of complications:
– Blindness
– Heart disease
– Kidney problems
– Sexual dysfunction
– Nerve damage

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 Glucose biosensor types
• In general, most of them are based on enzymatic sensing
approach, i.e. use enzymes to act as catalysts for chemical
reactions involving glucose.

• There are many different techniques:

– Optical techniques – non-Invasive
• Infrared/near-infrared spectroscopy
• Raman spectroscopy
Direct observation of glucose fingerprint using in vivo Raman spectroscopy Science Advances
24 Jan 2020: Vol. 6, no. 4, eaay5206 DOI: 10.1126/sciadv.aay5206

• Optical coherence tomography (OCT)

– Electrochemical technique – minimally invasive

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 Key components
• Blood lancet (or lancet), is a small scalpel but
with a double-edged blade or needle, that
are used to make punctures to obtain small
blood specimens, are generally disposable

• The electrochemical blood glucose test

strip (EBGTS) is a very small volume
(often about one µl or less) disposable
electrochemical cell, which is contacted
with whole blood.

• It then produces, in conjunction with a

test meter, an electrical current which is
proportional to the blood glucose
concentration. (Typical currents ~ few µA)

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 Basic mechanism
•Advance in direct measurement of blood glucose came in the late 1980’s,
when the enzyme glucose oxidase (GOx) became more widely available.

• Being an enyzme, GOx oxidizes glucose to gluconic acid, in doing so liberating

hydrogen peroxide (H2O2), which can then be detected electrochemically.

GOx
Glucose + O2 Gluconic acid + H2O2

What exactly happens here is…

Glucose + GOx GOx-H2 + Gluconic acid

GOx-H2 + O2 GOx + H2O2

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 Basic mechanism
Electrochemical detection of H2O2:

H2O2 O2 + 2H+ + 2e-

So the hydrogen peroxide current is proportional to the concentration of

hydrogen peroxide.

working reference
electrode electrode The roles of the anode:
A
(i) supplying electrons
(Pt) (Ag/AgCl) (conducting the circuit)
(ii) referencing potential

H2O2 H O Enzyme layer

2 2
(Gox)
Semi-permeable
Glucose + O2 membrane
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 First-generation Glucose biosensors
Glucose + GOx GOx-H2 + Gluconic acid
GOx-H2 + O2 GOx + H2O2

H2O2 O2 + 2H+ + 2e-

•H2O2 current as an indirect measure of glucose concentration

•Disadvantage: O2 concentration can vary in different samples

and fluctuate in times → a large error in measurements.

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 First-generation Glucose biosensors
Glucose + GOx GOx-H2 + Gluconic acid
GOx-H2 + O2 GOx + H2O2

H2O2 O2 + 2H+ + 2e-

•Oxygen’s only role is as an electron shuttle (via H2O2), removing electrons from the
GOx-H2, and taking them to the electrode.

Electrode
Glucose Gox H2O2
(substrate) (Enzyme [Ox])

Gluconic acid GOx-H2 O2 e-

(product) (Enzyme [Red])

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 Second-generation Glucose biosensors

•This problem could be by-passed by providing an artificial electron shuttle,

which transports electrons more effectively than O2 and H2O2
Fe(C5H5)2.
• Such artificial electron shuttle is called mediator, e.g. Ferrocene derivatives,
ferricyanide, conducting organic salts, transition metal complexes
[Fe(CN)6]3−
Electrode
Glucose Gox Mediator
(substrate) (Enzyme [Ox]) [Red]

Gluconic acid GOx-H2 Mediator e-

(product) (Enzyme [Red]) [Ox]

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 Second-generation Glucose biosensors

5-10 nm

Flavin adenine dinucleotide (FAD)

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 Second-generation Glucose biosensors
Schematic of enzyme immobilized within redox polymer
with arrows indicating electron transfer from glucose to
the working electrode via enzyme and mediator.
ECS Sensors Plus, 2022 1 031601
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Electrons diffuse in the redox hydrogels by collisions
(not hopping) between mobile redox centers
tethered to backbone of the cross-linked and
hydrated polymer. When the tethers are long enough,
the reduced (blue) and the oxidized (red) members
of the couple can come within the Marcus theory-
defined distance d that the electron can cross.
Current Opinion in Chemical Biology 2006, 10:664–672
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 Second-generation Glucose biosensors

where n is the number of electrons transferred per molecule

of the reactant, F the Faraday constant, T the temperature
Icat / ip,a (K), R the gas constant, cz is the substrate concentration,
k the rate constant, σ the stoichiometric coefficient, and ν
is the scan rate.
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 Other-generations Glucose biosensors

Citation: ECS Sensors Plus, 2022 1 031601

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 How to extract the current? Electrochemical cell
(Pt) A
Working (Ag/AgCl)
electrode Counter electrode

H2O2 H O Enzyme layer

2 2
(Gox)
Semi-permeable
Glucose + O2 membrane

This “two-electrode” configuration is unstable.

• If a current is flowing across a solid-electrolyte interface, chemical reactions will

occur at the interface.
• Due to the chemical reactions at the interface, the composition of the electrolyte
near the surface of the solid will change continuously and thus the distribution of the
voltage across the electrode-electrolyte interface will also vary.
→ It is difficult for the working electrode to maintain a constant potential while
passing current to counter redox reactions at the working electrode.
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 Standard Electrochemical setup :
Three-electrode cell
•What we want is to investigate the redox properties of only the species at the working
electrode (WE), we must circumvent this problem by preventing any current from
passing through the reference electrode.
•Solution: To divide the role of supplying electrons and referencing potential into two
separate electrodes.
•Reference electrode (RE)
•to act as reference in measuring and
V A controlling the working electrodes potential
WE RE CE and at no point it passes any current.

•Auxiliary electrode or Counter electrode (CE)

• to complete the cell circuit.
RE
WE CE

The above circuit doesn’t tell us how to maintain the voltage across WE and RE …
This is done by a device called “Potentiostat”
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 Potentiostat
Potentiostat
A
It controls the voltage difference between a CE
WE
working electrode and a reference electrode V
by negative feedback mechanism. RE

Control amp.
+
Vs – CE
RE

WE

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 How does it perform the feedback?
Let’s look at this simplified equivalent circuit,
Interface impedance of
+ V1
the CE and solution
resistance between the
– ic CE and the RE.
First, Control amp. equation: Vs
Z1
V1 = A (VS − VR ) (1)
VR

Interfacial impedance of
Z2 the WE and the solution
resistance between WE
and RE.
The op-amp output current can be written as:
V1 Combining (1) and (2):
iC =
Z1 + Z 2
Z2 VR 1
VR = V1 = V1 (2) =
iC =
VR Z1 + Z 2 VS 1
Z2 +1
A

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 Current-to-voltage converter
Control amplifier

+ Vout = −iE R f
Vs – CE Rf
RE
iE
WE – Vout
+

Current follower

Suppose feedback resistor Rf= 1MW and iE=1.0 mA ➔ Vout= - 1.0 V

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 Electrochemical cell used in
blood glucose biosensor
•Working electrode – an inert material,
such as carbon, gold, or platinum.

•Counter electrode and Reference

electrode – a mixture of silver and silver
chloride (Ag/AgCl).

blood glucose test strip 88

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 So..how does the measurement result
look like?
A B C D
Current Current Current Current

Time Time Time Time

• All EBGTS produce a time varying current which must be analyzed to

deduce a glucose concentration.
• Method of analysis varies substantially, depending upon the strip design.
Two main approaches:
• Amperometric - the instantaneous current at a particular time is
proportional to glucose
• Coulometric - the integrated charge underneath the entire curve is
proportional to glucose. 89
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 Continuous Real-time Glucose Monitoring Systems
• A continuous blood glucose monitor (CGM) provides maximal
information about shifting blood glucose levels throughout
the day (every few minutes) and facilitates the making of
optimal treatment decisions for the patients.

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 Glucose Monitoring - CGM
A typical CGM consists of:
•a disposable glucose sensor
•a link from the sensor to a transmitter;
•an electronic receiver that displays blood glucose levels as well as rising
and falling trends in glycemic (glucose level) excursions.

Abbott FreeStyle Medtronic MiniMed Paradigm® DexCom™ SEVEN®

Navigator® REAL-Time PLUS
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 Measuring glucose in interstitial fluid
• Tracks blood glucose levels by measuring glucose
concentration in interstitial fluid of subcutaneous tissue.
• Subcutaneously implantable devices
– composed of a microelectrode with a thin coating of glucose
oxidase beneath several layers of biocompatible membrane.
– continuously converts glucose from patient’s interstitial fluid
into electrical signal.
– commonly designed to operate for a few days, after which
they are replaced by the patient.
• Commonly inserted into the subcutaneous tissue in the
abdomen or upper arm.

Detailed measurement can be referred to : J Diabetes Sci Technol. 2010 ;4(5):1087-98. 92

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 Example: Abbott FreeStyle Navigator®

Sensor inserter

Sensor mount

93
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 Example: Abbott FreeStyle Navigator®

http://www.abbottdiabetescare.co.uk/ 94
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 Example: Abbott FreeStyle® Libre
Abbott FreeStyle® Libre

People using CGM system spent 38 % less time within hypoglycemia as compared with
those who managed their glucose with traditional self-monitoring glucose system.

https://www.abbott.com/corpnewsroom/diabetes-care/revolutionizing-cgm-with-freestyle-libre.html

Clinical study: “Bolinder, Jan, et al. Novel glucose-sensing technology and hypoglycemia in type 1 diabetes: a 95
multicentre,
MEDE 4500 non-masked, randomised controlled trial. The Lancet 388.10057 (2016): 2254-2263
 Another example:
Medtronic MiniMed

https://www.medtronicdiabetes.com/treatment-and-products/minimed-530g-
diabetes-system-with-enlite

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 Insulin Delivery Modes
Insulin Pens/Devices
•An easy-to-use, convenient, and accurate method
of insulin delivery.

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 Insulin Delivery Modes
Insulin Pumps

The prototype of the first pump that Omni Pod - the world’s first tubing-free insulin pump.
delivered glucagon as well as insulin,
backpack style, was in the early '60s.
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 Insulin Delivery Modes
Insulin Pumps
• Provide continuous insulin delivery
• Infusion site needs to be changed only every 2-3 days
• More reliable, precise insulin action
• Fewer missed doses
• Less insulin stacking
• Fewer social limitations
• Better data access for providers and patients

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 Closed Loop – CGM →Insulin pump
• Still needed:
• Faster insulins
• Better CGM accuracy
• Less sensor lag time

• Glucose control algorithms that

won’t fail

• Closing the loop will come in

small steps over time
Long-acting insulin is a slow-release insulin that works
throughout the day to maintain a baseline insulin level.
Rapid-acting insulin is used to cover meals and prevent
a sharp rise in blood sugar after eating.
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 Blood glucose meter interfaced
with mobile devices
Dexcom G6
GlucoPhone

Verio ®
iBGStar®

2008 2012 2016 2020

• Interface meter with iPhone via wireless (Bluetooth)
• Track BG data intermittently or continuously
• Will it be a game changer? Does it make people want to check their blood sugars more often
and that will lead to better health
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 Thank you !

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