G. Rosati BioDevices Labssie.dei.unipd.it/wp-content/uploads/2018/07/SSIE... · 1. Biosensors...

Biosensors and their

applicationsG. Rosati – BioDevices Lab

Summer School of Information Engineering SSIE 2018

Brixen

Biosensors and their

applications summary

23/07/2018 Biosensors and their applications 2

1. Biosensors fundamentals and market

2. Fabrication and characterization techniques

3. Electrochemical biosensors case studies: I. Microelectrodes for cells adhesion and

electroporation

II. DNA hybridization low-cost sensors

III. Multipurpose electrochemical lactate detection

IV. Inkjet printed sensors for antibiotics and phages

detections

1. Biosensors fundamentals

and market: sensors


Following the Oxford English Dictionary

definition, a sensor is a device that detects or

measures a physical property and records,

indicates or otherwise responds to it.


and market: sensors



and market: BIOsensors


The analyte is the specific target substance or chemical

constituent of the sensor detection.

A transducer converts an observed event, physical or

chemical, into a measurable signal, whose magnitude is

proportional (related) to the concentration of the analyte.

A biosensor is a device for the detection of

a specific analyte that combines a

biological element with a transducer.



and market: BIOsensors


Grand View Research, 2015

The biosensors impact on the society


and market: market

923/07/2018 Biosensors and their applications


and market: market

Biosensors in the medical field

1023/07/2018


and market: marketBiosensors revenue by application

A. Antoniacci - The global biosensor market incomes from specific detection application (2009).



and market: definitions

Selectivity: is the ability to discriminate between different

substances (analytes).




Sensitivity: Is the minimal detectable analyte

concentration change.

IUPAC (International Union of Pure and Applied Chemistry)

defines sensitivity as the slope of the linear part of the calibration

plot.




Accuracy: Is the degree of

closensess of

measurements of a quantity

to the quantity’s true value.

Precision: Is the degree to

which repeated

measurements under

unchanged conditions show

the same results.




Response time: Time needed by the sensor to produce

a stable response.

Recovery time: Time that elapses after a measurement

before the sensor is ready to analyze the next sample.

Working lifetime: Time during which the sensor can be

used maintaining its characteristics. It’s usually

determined by the stability of the selective material.



and market: bioreceptors

The recognition elements are the key

component of any sensor device. They impart

the selectivity that enables the sensor to

respond selectively to a particular analyte or

group of analytes, thus avoiding interferences

from other substances




The biological recognition elements of a biosensor perform the

following two key functions:

1. It specifically recognizes the analyte.

2. It interacts with the analyte in such a manner, which produces some

physical change detectable by the transducer.

Recognition elements can be:

• catalytic activity

• high sensitivity

• high selectivity

• linear response

• requires enzyme purification

Enzymes










• high sensitivity


• sandwich/competitive immunosensors

• long time response

Enzymes

Antibodies










• exploit DNA properties


• stability

• long time response

• short sequences

Enzymes

Antibodies

DNA










• use modified cells

• variety of enzymes

• contaminations

• low selectivity

Enzymes

Antibodies

DNA

Whole cells or tissues










• low cost


• variety of aptamers

• high reproducibility and stability

Enzymes

Antibodies

DNA

Whole cells or tissues

Aptamers


2. Fabrication and

characterization techniques

There exists a wide variety of fabrication methods and even more

characterization techniques in the biosensors field.

In this lecture, we will present some of the most relevant for the

production and characterization of electrochemical biosensors:

FABRICATION

Photolithography (microfabrication)

Screen-printing

Inkjet printing

CHARACTERIZATION

Electrochemical Impedance

Spectroscopy

Cyclic Voltammetry


2.1 Fabrication techniques:

photolithography

Photolithography is a subtractive fabrication process where the

selective removal of material allow to obtain well resolved 3D shapes.



photolithography

Clean room fabrication allows to obtain nanostructured surfaces for specific

and sensitive materials interactions with biological molecules.

Manipulation of the matter at the atom level!



photolithography

Ref.

FE

T

Clean room fabrication allows to obtain nanostructured surfaces for specific

and sensitive materials interactions with biological molecules.



photolithography

Axon/dendrites action potential propagation Nanohole FET DNA sequencingF. Patolsky et al, Science 313 (2006) 1100. Xie et al, Nature nanoterchnology vol. 7, pg 119-125 (2011)

Screen-printing is an additive technique for the fabrication of thick-film and

low-cost devices.



screen-printing


low-cost devices.



screen-printing

This technique is employed

also in industrial productions,

e.g. t-shirts, CDs/DVDs printing.

A different mesh

(screen) is required

per each color

(layer)


low-cost devices.



screen-printing

Electrochemical sensors

can be produced by using

conducting and insulating

inks on rigid or flexible

substrates.



screen-printingScreen-printing is an additive technique for the fabrication of thick-film and

low-cost devices.

Smart tattoo for the stress level electrochemical

quantification (sweat sample)Glucose monitoring

strips (blood sample)



inkjet printingInkjet printing is an additive technique for the fabrication of thin-film and

low-cost devices. It can be based on:

• Thermal inkjet

• Piezoelectric inkjet

Differently from screen-printing, this technique does not require any

preparation process before printing

Thermal inkjet Piezoelectric inkjet




low-cost devices. The devices can be produced both by consumer and

research-grade printers.




low-cost devices.

Moya et al. SPIE Microtechnologies 2017




low-cost devices.


2.4 Detection techniques: Electrochemical Impedance Spectroscopy

Electrochemical Impedance Spectroscopy (EIS) studies the electrode

surface by means of its IMPEDANCE.

• Possibility to detect transducer surface binding events, surface properties

changes and modifications far from the surface too.

• Impedance spectroscopy is a non-destructive technique and so can provide

time dependent information about ongoing processes.





NO redox

mediator





WITH

redox

mediator

Randle’s model





WITH

redox

mediator10

110

210

310

410

510

3.1

103.4

103.7

|Z| [

]

101

102

103

104

105

-30

-25

-20

-15

-10

-5

[°]

Frequency [Hz]

Rct + Rs Rs

1/Cdl



The DC bias potential is fundamental to observe the charge transfer

resistance and depends on the particular redox mediator (E0) and on the

electrodes’ materials used.

0

2

4

6

8

10

12 0

100

200

300

400

5006

7

8

9

10

11

12

Bias potential [mV]

log(Frequency) [Hz]

Rct + Rs

Rs

1/Cdl

Lo

g(|

Z|)

[lo

g(Ω

)]


2.4 Detection techniques: Cyclic Voltammetry

Cyclic Voltammetry (CV) studies the behavior of redox mediators on the

electrodes and can detect/induce their oxidation (reduction).


2.4 Detection techniques: Cyclic Voltammetry

Cyclic Voltammetry (CV) studies the behavior of redox mediators on the

electrodes and can detect/induce their oxidation (reduction).

Input potential

CV are dynamic measurements and relies on diffusion, electrooxidation

(reduction) of mediators, thus they are difficult to model but offer a very

sensitive way to measure the electrode’s surface characteristics and to

detect electroactive molecules.

Case study IMicroelectrodes for cell adhesion

and electroporation

4223/07/2018

3.1 Case studies: microelectrodes

for cell adhesion and electroporation

The goal of this work was to monitor the electropermeabilization of

adherent mammalian cells through electrochemical impedance

spectroscopy.

• Electropermeabilization or

electroporation (EP) is the

phenomenon where the cell

membrane’s pores opens

in response to an electrical

stimulation.

• The pores opening can be used to introduce

chemicals in the cells, i.e., genetic material to

induce its mutation or to use it for the production

of specific proteins.

A. Ferrario et al. 2011, 7th Conference on PhD Research in Microelectronics & Electronics, pp. 57-60.

23/07/2018

3.1 Case studies: microelectrodes


Cells plating on microelectrodes array (MEA) and EIS experiments showed

4 impedance groups related to the number of cells over each electrode.


45Biosensors and their applications

3. Case studies: microelectrodes


EIS measurements fit with the electrical model showed good results and

dependence of Rseal and β by the number of cells on the electrode.


46



The evaluation of electroporation effects was performed by measuring EIS

immediately before and after the cells electrical stimulation (achieved by

the same electrodes).

As a control, a fluorescent dye was added

in the cells solutions and the electroporated

cells were compared to the not stimulated

ones for the dye inclusion.

23/07/2018

E. Pasqualotto et al. 2012, Procedia Chemistry, vol. 6, pp. 79-88.

47






23/07/2018


48








49



23/07/2018

From the EIS measurements fit is possible to obtain also the average

number of opened pores and an evaluation of their radius.

Potential (V) Poresnumbers

0.3 0

0.6 0

0.9 0

1.2 500

1.5 1300

1.8 2500

2.1 4500

2.4 6000


Case study IIDNA hybridization thin-film

sensors


3.2 Case studies: DNA

hybridization thin-film sensors

DNA hybridization is the matching of single-sided DNA (ssDNA)

complimentary chains. DNA hybridization sensors are used to check if a

specific DNA fragment is present in a sample.

• Array of microwells for the

hybridization of different

ssDNA chains, called

microarray, are commonly

used for multiplexed

detection.

• Microarrays are based on

optical fluorescence

response, thus they require

expansive instrumentation.

5223/07/2018



An electrochemical alternative to microarrays could help to bring the

hybridization sensors to the point of care reducing the instrumentation

costs.Low-cost devices produced engineering the

CD/DVD fabrication process with a patented

method:

Polycarbonate injection moulded

(nanostructured) substrate.

Thin-film gold patterned flat layer (sputtering,

50 nm).

Screen-printed UV-cured patterned insulating

layer.

A. Ferrario et al. 2012; Procedia Chemistry, vol. 6, pp. 36-45.

5323/07/2018



An electrochemical alternative to microarrays could help to bring the

hybridization sensors to the point of care reducing the instrumentation

costs.

MCH: 6-Mercapto-1-hexanol

(SH-(CH2)6-OH)


5423/07/2018

3. Case studies: DNA hybridization

low-cost sensors

EIS measurements were performed in presence of redox mediator. The

results were fit with the standard Randle’s model to obtain the charge

transfer resistance values.

With this method is possible to study both the electrode surface coverage

by ssDNA probes and to detect the complimentary ssDNA hybridization.


5523/07/2018



EIS measurements in presence of redox mediator for the study of the

immobilization of ssDNA/MCH mix with 1:1 and 1:10 ratios.

0 20 40 60 80 100 120

104

105

Time [min]

Ch

arg

e t

ran

sfe

r re

sis

tan

ce

Rct [

]

2 M ssDNA/2 M MCH

2 M ssDNA/20 M MCH


5623/07/2018



Probe surface density for hybridization detection optimization.

0.002 0.02 0.2 2 2010

3

104

105

106

MCH concentration [M]

Ch

arg

e t

ran

sfe

r re

sis

tan

ce

Rct [

]

MCH

MCH/2 M ssDNA

MCH/2 M ssDNA/1 M target


5723/07/2018

3. Case studies: DNA hybridization

low-cost sensors

EIS detection of ssDNA hybridization requires the use of spacers

between the ssDNA probe chains in order to enhance the ssDNA target

detection sensitivity.

The system can be easily

implemented in a multiplexed

format by adjusting the device

layout.


Case study IIILactic acid sensing in cell

cultures, sport, and medicine

O2

Lactate

H2O2

Pyruvate

LOx

2e- + 2H+

NAD+

Lactate

NADH

Pyruvate

LDH

2e- + H+

5923/07/2018

3.3 Case studies: Lactic acid

sensing in cell cultures, sport, and

medicine

Lactic acid is a metabolite involved in many biological processes such

as hypoxia in clinical and sport applications, food spoilage by bacteria

contamination, and food fermentation industry (cheese, wine).

As glucose, lactic acid (or lactate) detection is usually performed by

enzymatic reactions, such as lactate oxidase (LOx) or lactate

dehydrogenase (LDH).

Results under publication




medicine

As glucose, lactic acid (or lactate) detection is usually performed by

enzymatic reactions, such as lactate oxidase (LOx) or lactate

dehydrogenase (LDH).

The enzymes react specifically with lactate and the products of their

reactions are the analytes to be detected by the sensors or by other

techniques.

UV-Vis spectroscopy Cyclic Voltammetry





medicine

The NADH detection can be performed on screen-printed electrochemical

biosensors by Cyclic Voltammetry and also measuring the current at a

fixed potential.





medicine

The NADH detection can be performed also optically, by its specific

absorbance peak at 340 nm. The LDH enzyme production of NADH in

presence of Lactate can be monitored at 340 nm in real-time.





medicine

The correlation between the optical and electrochemical determinations

of the NADH produced by the enzyme in presence of Lactate is high.

However, the efficiency

of the Lactate-NADH

conversion is lower than

2% and depends on the

enzyme isoform.

However, there are

strategies to drastically

increase the efficiency.

𝐸𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 = 100[𝑁𝐴𝐷𝐻]

[𝐿𝐴]




medicine

Rapid removing of pyruvate shifts the reaction equilibrium towards the

NADH formation, increasing the LA-NADH conversion efficency.

61Biosensors and their applications23/07/2018Results under publication

65



medicine

Biosensors and their applications23/07/2018

Use of the LDH-GPT protocol and of CV measurements on screen-

printed electrodes for the detection of LA produced by cell cultures

incubated over time in DMEM and DMEM+FBS at 37°C.


66



medicine


Use of the LDH-GPT protocol and of CV measurements

on screen-printed electrodes for the detection of LA

before and after exercise in mice plasma.


Biosensors and their applications

67



medicine


Use of the LDH-GPT protocol and of CV measurements on screen-

printed electrodes for the detection of LA in sweat during physical

exercise.


Case study IVInkjet printed biosensors for

antibiotic and viruses detection


3.4 Case studies: Inkjet printed

biosensors for antibiotic and viruses

detection

• Inkjet printing is familiar as a method of printing text and images onto

porous surfaces (Calvert, 2001).

• Since the 80s this technology has been increasingly used in research

laboratories around the world for the fabrication of whole (bio)chemical

sensing devices (Li, 2012).

• This technology provides distinct advantages over other fabrication

processes (Komuro et al.

2013), such as:

High speed/resolution

Low-cost

User friendly




detection

• Office inkjet printer loaded with commercially-available AgNP-based ink.

• Design and print of 100-500 µm wide AgNP lines

on office paper, glossy paper, and coated PET.

• AgNP lines morphological and electrical characterizations.

100 µm

200 µm

300 µm

400 µm

500 µm

Desig

nP

rint

Glossy photo paper

100 µm nominal width

~150

µm

~0,8 µm


Electrochemical Impedance Spectroscopy (EIS) measurements of the lines.

Admittance @10 Hz and linear fit with respect to measured width (Wmeas) for

sheet resistance (Rsheet) calculation.

71

*Required heating to 150-300°C

Substrate Rsheet (mΩ)

Glossy photo

paper112,7

Paper 8312,6

Coated PET 137,7

Kodak paper 56,9* (Shen 2014)

Glass 301,0*(Lee 2005)

Paper 696,0 (Määttänen 2012)

PET 467,0* (Ihalainen 2012)




detection





detection

Design, printing and test of interdigitated electrodes (IDE) for affinity

biosensing applications.

Development of a 6 channel workbench with

plug in connection.

Verification of electrodes dimensions for high

devices reproducibility.

Definition of paper devices resistance to

solution absorption and to multiple rinse (10).





detection

10-1

100

101

102

103

104

105

106 1 10 20 30 40 50 60 70 80 90

101

102

103

104

Time [min]

Impedence measurement of PBS Kinetics

Frequency

Ma

gn

itu

de

|Z

| [

]

IDE stability in 10 mM Phosphate Buffered Saline (PBS) buffer pH 7.4

Initial change of the

low frequency

impedance (Rct).

Drift of the solution

resistance due to

evaporation (Rs).

Slight drop of the

impedance after 1

hour (Rct).


Design, printing and equivalent

electrical circuit modelling in PBS

of interdigitated electrodes (IDE)

for biosensing applications. CdlRs

Rel (fixed) CIDE

wl




detection


Self Assembling Monolayers (SAM)

are molecules that autonomously

form monolayers on plain surfaces.

SAM are often used in order to

simulate binding events on the

electrode’s surface and to verify

their detectability by EIS, which

defines the electrodes quality for

the biosensor realization.

75

6-mercapto-1-hexanol (MCH) is a

SAM molecule which has a thiol head

group that covalently binds to metals.

Impedance kinetics at 10 Hz of 1 mM MCH binding to the IDE

10 mM MCH

PBS

1 mM MCH

in PBS




detection





detection

A topic that is attracting the public attention is the presence of

antibiotics in meat and milk, which reflects the abuse of

antibiotics in animal breeding.

These antibiotics presence is dangerous both in the short and

long-term perspective:

• In the short term it can cause adverse reactions like

allergies.

• In the long term the antibiotics abuse constitutes the base of

the antibiotic resistance development.

Ampicillin is one of the most diffused antibiotics

and aptamers for its specific detection are

commercially available.





detection

Test

electrodes

Aptamer

functionalized

Control

electrodes

NOT

functionalized

Ampicillin

spiked milk

tests





detection

Bacteriophages or phages are viruses that attack bacteria only,

replicating within them, and causing their death (Duckworth, 1976)

Cheese, yoghurt, and wine are

produced by adding bacterial

cultures (starter cultures) to the

unprocessed raw material, that

cause controlled fermentation

and acidification.

Lactic bacteriophages are one of the biggest problems for dairy and wineindustry because they stop fermentation processes, and lead to low quality products (in terms of nutritional values, taste and consistency) (Sillankorva et al. 2012, Jonczyk et al. 2011)

Phages

Bacteria

100 nm




detection

13/06/2018 28th Anniversary World Congress on Biosensors 79

IDEA layout and assembled system

Time (hours)

|Z| (Ω)

M17

Culture medium

(STABILIZATION)

M17+

Bacteria

(GROWTH)

M17+

Bacteria+

Phages

(ATTACK)

4 7 25

BACTERIA

INJECTION

PHAGES

INJECTION

//EIS START EIS STOP

Cycling of the EIS

measurements on the

channels by switch

matrix.


13/06/2018 28th Anniversary World Congress on Biosensors 80



detection

Full EIS spectra cycling over the 8 channels:o Channels 1-4: M17 + Bacteria + Phage buffer

o Channels 5-8: M17 + Bacteria + Phages

M17 + Bacteria + buffer

M17 + Bacteria + phages Selection of the 0.1 Hz

frequency for the

kinetics plots

Normalization of

impedance magnitude

and phase values:

With k1 and k2 chosen to

start from zero.

∆𝐵𝑎𝑐𝑡 = 𝑍𝑔𝑟𝑜𝑤𝑡ℎ − 𝑍𝑠𝑡𝑎𝑏 − 𝑘1

∆𝑃ℎ𝑎𝑔𝑒 = 𝑍𝑎𝑡𝑡𝑎𝑐𝑘 − 𝑍𝑠𝑡𝑎𝑏 − 𝑘2


Take home

messages

Take home messages:

Biosensors


Biosensors are devices which detects specific

analytes by combining a transducer and a

biological part.

Biosensors impact on the market will increase

in the next years in the medical, food, and

environmental sectors.

Biosensors’ performance can be compared by

assessing their specificity, sensitivity, accuracy,

precision, response/recovery times.

Take home messages:

Fabrication


Costs Prototypingtime

Processcomplexity

Resolution

Clean-room High High High +++

Screen printing Medium Medium Medium ++

Inkjet printing Low Low Low +

Clean-room Screen-printing Inkjet-printing

Inkjet printing Vs standard techniques for sensors production:

Take home messages:

Characterization


Electrochemical methods are just few of the

available characterization techniques in the

biosensors field.

EIS and CV are two of the most used

techniques between electrochemical methods.

EIS and CV allows the quantification of

electrodes surface coverage by biological

molecules.

Take home messages:

Case studies

23/07/2018 85

Monitoring of the electroporation of adherent mammalian cells through

electrochemical impedance spectroscopy is possible and more

efficient than fluorescence testing.


Take home messages:

Case studies

23/07/2018 86

DNA hybridization can be detected by EIS on thin-film low-cost sensors

and the detection efficiency depends on the ssDNA probe density.


Take home messages:

Case studies

87

Lactic acid can be detected by screen-printed electrochemical biosensors

in cultures medium, plasma, and sweat with an efficiency over 70%

using a two-enzyme protocol.


Take home messages:

Case studies

Inkjet printed biosensors can be used to detect Ampicillin by aptamer

functionalization and phages by bacterial culture impedance monitoring

over time.


Thank you for your

attention!


BioDevices Lab.http://biodevices.dei.unipd.it/

Prof. Alessandro Paccagnella

Giulio Rosati, PhD

ARC

Centro Ricerche Applicatehttp://www.arc-projects.it/

Alessandro De Toni, PhD

Elisabetta Pasqualotto, PhD

Matteo Scaramuzza, PhD

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