UG Project Report

NANO - MATERIAL BASED BIOSENSORS

AJAY MAHICH (17014003)

A report submitted in part fulfilment of the degree of

IDD (B.Tech+M.Tech) in Biochemical Engineering

Supervisor: Dr. Pranjal Chandra

School of Biochemical Engineering

Indian Institute of Technology (BHU)
Varanasi
 Acknowledgement

I would like to express my incredible Thanks of gratitude to my

Professor Dr. Pranjal Chandra who gave me the golden chance to do

this amazing project on the topic “Nano – Material Based Biosensors” . I

came to recognize about so many new matters . Then I would like to

thank my friends who have helped me with their valuable pointers and

comments which was very valuable all through quite a number phases of

the project completion .

 Index
 Abstract

 Introduction

 Materials

 Methods

 Conclusion

 Refrence
ABSTRACT :

In this report we have discuss about nano-material based biosensors .

Here we are using as nanomaterial : AuNPs (gold nano-particles) . We

characterize gold nanoparticles using UV–

Visible absorption spectrophotometry , dynamic light scattering (DLS) ,

transmission electron microscopy (TEM) and raman sprectroscopy . We

are using gold nanoparticles in biosensors because gold nanoparticle

are increased biosensor detection signal, or response, for analytes that

are especially low in concentration . Then we classified gold

nanoparicles based biosensors .

INTRODUCTION :

BIOSENSOR :

Biosensor is an device that detects specific bioanalyte . Biosensor is an

sensing device used for estimation of biological material (material like :

antibody , cells , dna ) by biological interactions and this intraction

produce physiochemical changes which is detected by transducer and

convert into signal , this signal is interpreted and converted to bioanalyte.

Biosensor is to provide rapid , real time , accurate information about the

analyte . biosensor is utilize the high sensivity and selectivity of

biological sensing . Biosensors are used for food quality estimation,

environmental monitoring, and diagnosis of clinical and metabolic

complications have come to the fore.

Nanomaterials :

Nanomaterial are the basically very small size particles . These are

generally found between 1-100 nm range . These are divided into two

parts – nanostructured material and nanoparticle materials . Nano

material can be made up of metals , polymers , organic or inorganic

materials etc. Nanomaterials can be synthesized by physical , chemical

and biomedical process . These are basically divided into top down and

bottom up methods . In top-down process size reducing of the bulk

material and In bottom up process material synthesis from the atom level

. when we do synthesize nanoparticles then we need to control particle

size distribution , purity , quality , quantity and morphology etc . In Our

process we use as a nanomaterials AuNPs (Gold nanoparticle) . AuNPs

is the diameter of 1-100 nm size partcles and they have higher surface

volume ratio and higher surface energy to provide a stable

immobilization of a large amount of biomolecules. AuNPs are Able to

fast and direct electron transfer between a wide range of electroactive

species and electrode materials. AuNPs Used in biomedical and

environment monitoring applications due to their excellent optoelectronic

and increased physico-chemical properties. Gold nanoparticles can be

simply synthesize because they are colloidally stable and they can
directly combined with biological molecules. Due to their optical

properties ( Quantum effect of light on material used on electronic device

) , in particular the surface plasmon resonance ( electron oscillation on

gold nanoparticle plate) they can be visible with different methods and

sensors based on changes of the plasmon resonance have been

exhibited . Metal Nanoparticles are generally used as components of

electronic wires.
Materials :

Hydrochloric acid (HCl), Deionized water , Chloro‐auric acid (HAuCl4),

Trisodium citrate ( Na3C6H5O7 ) , Membrane filter paper .

METHODS :

PREPARATION OF AuNPs solutions :

HAuCl4 solution with the concentration of 0.49 mol/L is prepared by

dissolving 500 mg of HAuCl4 into 3 mL of 10% HCl.

Then, a diluted 0.2 mM of HAuCl4 solution is prepared by adding 40 μL

(19.6 μmol) of HAuCl4 solution into 100 mL of deionized water. Then we

get 2 solution as a products :

Solution A - 558.79 mg of trisodium citrate is added into 50  mL of

deionized water .

Soution B - The concentration of the solution is controlled at 38.8 mmol/L

and Solution A takes into a rolling boil (liquid boil rapidly with many

bubbles ) at 150°C with stirring vigorously to got homogenous size

(uniform or same in size) of the AuNPs solution.

10 mL of 38.8 mM of sodium citrate is added quickly into the vortex of

the solution. Then we get a color change solution from pale yellow to

red. Boiling and stirring are continued for another 10 min. Then heated

and then removed, and stirring continued for an additional 15 min. When

the solution cooled down to room temperature, it is filtered by 0.8 μm

membrane filter paper. Then final solution keeps in the refrigerator with
temperature 4°C and measured by using UV spectrophotometry at

wavelengths between 400 nm to 800 nm.

Characterization Techniques of Gold Nanopaticles

(AuNPs) :

AuNPs are characterized by some techniques UV–

Visible absorption spectrophotometry , dynamic light scattering (DLS) ,

transmission electron microscopy (TEM) and raman sprectroscopy .

Dynamic Light Scattering (DLS) :

DLS is an analytical technique using for identify the particle size and

particle size distribution (range between nm to micro meter). In this

method detects the scattered light intensity variation caused by the

Brownian motion of the particles suspended in a liquid. We performed

DLS measurements of citrate stabilized AuNPs with diameters of 10, 20,

40, 60, 80, and 100 nm.

Transmission electron microscopy :

Transmission electron microscopy used for the idenfication of shapes

and size.This method is performing for using a well spreaded solution of

gold nanoparticles. This method is focus on the to thin beam of electrons

(stream of electrons that moving on same direction) onto a sample. The

beam of electrons moving through the sample and depending on the

density of the sample present, some of the electrons are scattered. The

unscattered electrons hit a fluorescent screen at the bottom of the

microscope and give rise to a image of the sample.

Raman spectroscopy :

Raman spectroscopy is a light scattering technique . In this method the

frequency of the photons (single unit of light) maybe change depending

on the sample interaction. The laser beam is an oscillating

electromagnetic field which depends on thr interaction with the

molecules and then they can deform it. Because of this deformation ,

molecules start vibrating with a specific frequency, which is subsequently

measured . Gold nanoparticles increases the Raman intensity this

increasement based on the particle size and environment.

UV - Visible Spectroscopy :

This method used to estimate the absorbance spectra of solution .gold

nanoparticle’s growth based on the absorption intensity of gold

nanoparticle size.UV-visible absorption spectra of gold nanoparticle are

estimated by uv spectrophotometer .Then the solution are taken at

different reaction time intervals. Deionized water used as a reference

sample. The morphology and the size of gold nanoparticles is prepared

by citrate synthesis .
Gold Nano-particle Based Biosensors :

Gold Nano-particles Based Electrochemical Biosensor :

Electrochemical biosensors are sensing devices that are produced by

modifying the electrode surface with biological molecules , like enzymes,

antibodies, and DNA.Gold nanoparticle-modified electrode surfaces can

be produced in three types:

(a) by binding the AuNPs with functional groups of self-assembled

monolayers (SAMs);

(b) by direct deposition of AuNPs onto the bulk electrode surface;

(c) by incorporating colloidal gold into the electrode by mixing the gold

with the other components in the composite electrode matrix.

Biosensors can be made by immobilization the biological molecules by

adsorbing upto the nanoparticles, by cross-linking them with bifunctional

agents (organic molecule with 2 different functional groups) like

glutaraldehyde, or by mixing them with the other components of

composite electrodes.

Biosensors are widely used in biomedical analysation , environmental

monitoring , control processing in food manufacturing

industries.Electrochemical biosensors can be used for estimating the

target molecule in sample solutions without pre-treatment of samples

owing to the specific binding or interactions between biomaterials and

target molecules.

Gold nanoparticles (AuNPs) shows high conductivity, high affinity and

biocompatible for biomolecules .

Gold nanoparticle-based electrochemical biosensors has been

constructed enzyme electrodes, immunosensors and DNA sensors .

Gold Nanoparticles Based Optical Biosensor :

Optical biosensor is an detecting device that including a bio recognition

element integrated with the transducer optical system.

The purpose of the optical biosensor is to provide signal and this signal

corresponds to bioanalyte filtering.an optical biosensor used in many

biological materials such as enzymes, antibodies, antigens, receptors,

nucleic acids, whole cells and tissues as biorecognition elements.

Surface plasmon resonance (SPR), evanescent wave fluorescence and

optical waveguide interferometry use an evanescent field near the

biosensor surface to detect the combination of biorecognition and

analysis.

The signal amplification pathway of GNPs in general can be shortened

to two points:

(i) electronic junction between localized with GNPs and which distributes

plasmon to the surface of the SPR and

(ii) maximum mass and mass of the high molecule of GNPs increase the

analytical weight of analysts immobilized them.

Gold nanoparticles absorb and deflect light with exceptional efficiency.

Gold nanoparticles have strong ineractions with light, since the electrons

on the metal region through collective osillation when they have excited

by light at specific wavelengths. This oscillation is called surface

plasmon resonance (SPR) and absorption and scattering intensity of

gold nanoparticles to be higher than the size of non-plasmonic

nanoparticles.

Modification of the surface is easy because of good affinity for Thiol and

amine groups in gold . Gold nanoparticles has a large absorption in the

visible and near-infrared range due to the stimulation of localized surface

plasmon resonance. When the intense light focuses on the gold

nanoparticles , the large optical absorption will increase the temperature

of AuNPs. The thermal effect of AuNPs can be used for phototherapy.

Gold nanoparticles images are determined by simply adjusting

illumination conditions for the bright field and PEW images. PEW is

generated on a glass slide. AuNPs near the surface of glass , giving an

very low background nanoparticle image. PEW setup has several

advantages over the standard total internal reflection (TIR) method. It

has same distribution of evanescent field. The depth of PEW is longer

and more suitable for observation of AuNP images . AuNPs images are

obtained by simply adjusting illumination conditions for the bright field

and PEW images. The evanescent waves coming from the surface of

overlayed fiber create an interactive field and only Fluorescent signals

of fluorophores are significantly increased within the interactive region

Receiving A biosensing system with high-sensitivity, local-area

enhancement of metal-nanoparticles More recently it has been widely

used due to surface plasmon excitation Fluorescence enhancement from

localized plasmon (LSP) resonance of nanoparticles was incorporated

into the biomolecule. fluorescence excitation using evanescent light.

evanescent biosensors has three main components: : (1) optics,

including waveguide, fluorescence light source and signal measuring

detector. (2) sample delivery fluid and reagents in waveguide.(3)

Electronic and computer processors for recording and detecting the

signal and controlling fluid.

Gold Nanoparticle Based Piezo-Electric Biosensor :

In Piezoelectric biosensor mostly used quartz crystal microbalance

(QCM) form , which is a sensitive technique based on the piezoelectric

effect . QCM biosensors are high sensitive at nanogram level and real-
time monitoring . QCM used in the sensing of genes, proteins, cells,

microorganisms, toxins. Due to QCM big specific surface area of AuNPs

are immobilized on the surface of quartz crystal with more connected

biological molecules in QCM biosensors. Gold nanoparticles are

immobilized on the surface of a AuNPs electrode to increases the no. of

probes and hybridized more target biological molecules . Sensitivity of

QCM method is 3 times more than without AuNPs

immobilization.Deposited AuNPs on the surface of platinum coated QCM

(Pt-QCM) to gives more binding sites for biological molecules. Gold

nanoparticles have a significant signal amplification effect in QCM

biosensors due to their greater mass more than biological molecules.

CONCLUSION :

In this report we knows about the nanomaterial based biosensors . We

are using as a nanomaterials : gold nano particles (AuNPs) because

unique optical properties and inertness make AuNPs perfect platform to

fabricate biocompatiblesensors . The optical properties strongly depend

on particle size, shape, composition and surface modification. We are

characterized gold nano particles using some characterization

techniques UV–Visible absorption spectrophotometry , dynamic light

scattering (DLS) , transmission electron microscopy (TEM) and raman

sprectroscopy . We are classified nanomaterial based biosensor in 3

parts : GNP-based optical, electrochemical and piezoelectric biosensors.

REFERENCES :

https://link.springer.com/article/10.1007%2Fs13205-019-1577-z
https://link.springer.com/protocol/10.1007%2F978-1-60327-569-9_11

https://www.sciencedirect.com/science/article/pii/S0013468608003599?via

%3Dihub

https://scihub.tw/https://www.worldscientific.com/doi/abs/10.1142/S179398

4416420010

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315477/

https://link.springer.com/article/10.1007%2Fs13205-018-1148-8

https://education.mrsec.wisc.edu/citrate-synthesis-of-gold-nanoparticles/

https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Book

%3A_Physical_Methods_in_Chemistry_and_Nano_Science_(Barron)/04%

3A_Chemical_Speciation/4.03%3A_Raman_Spectroscopy
https://www.sciencedirect.com/science/article/pii/S0013468608003599?via

%3Dihub

https://www.ingentaconnect.com/content/asp/jbn/2014/00000010/00000010/

art00012

https://www.jove.com/science-education/5795/optical-biosensing

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986466/#:~:text=An

%20optical%20biosensor%20is%20a,a%20measured%20substance

%20(analyte).

https://nanocomposix.com/pages/gold-nanoparticles-optical-properties