nanoengineered biosensors presentation srivastava aug 24 2011 · 2011-11-17 · srivastava and...

INDIAN INSTITUTE OF TECHNOLOGY, BOMBAYDEPARTMENT OF BIOSCIENCES AND BIOENGINEERING

Nanoengineered BioSensors

Rohit Srivastava, PhD

Email: [email protected]

Aug 24th 2011

mailto:[email protected]

OVERVIEW OF TECHNOLOGY NANOSENSORS GLUCOSE BIOSENSOR UREA BIOSENSOR NANO-in-MICRO

Motivation & Applications

• Particles for Biochemical

Analysis

– In vivo, culture, etc.

– Clinical or research

• Fiber Probes

– Ultrathin film coatings on tips

– Sensing probes

• Cell culture scaffolding

– Sensing of cell environment

(Kopelman, UM)

Wavelength (nm)

No

rma

lize

d In

ten

sity

(Walt, Tufts U)


Coated Templates Filled Capsules

Scheme A

Scheme B

Scheme C

Scheme D

Polyanion

Polycation

PolyionIndicatorReference Dye

Enzyme Labeled Protein

Labeled Ligand

Sensor Configurations

McShane, IEEE Sensors, 2002.


Layer-by-layer Self-Assembly

• Driven Primarily by Electrostatic Attraction

– Adsorption of charged species

– Saturation & reversal of surface charge

– Approximate monolayer growth (1-30nm)

{Polycation/Polyanion} × n;

n = 1 – 200

+

+

+

+

+

+

_

_

_

_

_

_

_

_

_

_

2

_

_

_

_

_

_

31

-

-

--

+

+

+

+

+

+

+

+

+

+

+

1

-

-

--

+

+

+

+

+

+

+

+

+

+

+

_

_

_

_

_

3

+

+

+

+

+

+

_

_

_

_

_

_

_

_

_

_

_

2

Polyion Assembly Assembly of Particles

{Polyion/enzyme} × n OR{Polyion/silica nanoparticle} × n


Multilayer Building Blocks

Polymers

- - -

- - -

- - -

- - -n

COO-

+++

+++

+++

+++NH2

+

nProteins, Enzymes, Viruses

Albumin, GOx, ...

(>20 proteins)Particles

SiO2, TiO2, Au, Clay, ...

•Any solid surfaces

•Micro/nanotemplates

•Porous Filters

Substrates:Dyes

300nm300nm

450nm

Nanoparticles can be deposited on

nanotemplates, e.g. silica on latex. Separation is

KEY.

>20 linear or branched polyions

Absorbers, Fluorophores


Advantages

• Advantages

– Deposition on surfaces of almost any kind and

any shape

– Many control parameters:

• Concentration

• Adsorption time

• Ionic strength

• Solvent composition

• temperature


Polyelectrolyte Capsules

• Latex or silica nanoparticles

serve as template

• Thin films assembled on surface

• Inner core removed (acid)

• Polyion ―shell‖ acts like cell

membrane

Confocal & AFM images

5 m shell, 20nm wall (PSS/PAH)4

Shells Produced by Core Removal

Template Dissolution Fragment

migration

5 m

5 m

Nanocapsule from SiO2 core

Fang, Grant, McShane, Lvov, Langmuir, 2001.


Template-loaded Micro/Nanocapsules

• Hydrogel entrapment of

functional molecules

• Nanofilm assembly on

polymer surface

– Stabilization of

entrapment

Ionically-crosslinked alginate templates + {Cresol Red-PAH/PSS}2 coating

Sodium

Alginate

w/ FITC-

Dextran

Harvested Spheres

after centrifugation

Overhead Stirrer

@ 5000 rpm

Oil Phase with

surfactants

Gelling Agent

Gelled

Microspheres

in Emulsion

Srivastava and McShane, Journal of Microencapsulation, 2005.


Unique Characteristics

• Internal reference

– Insensitivity to drift, environment

• Precise nanoconstruction

– Tailored optical, chemical, mechanical properties

• Complex architecture, simple production

– Many materials, novel properties

• Potential for complex reactions

– Multiple sequential reactions


Competitive Advantages

• Reliable, repeatable, precise methods

• Allows for unique functions

• More efficient use of time and resources

• Low cost, simple


“Smart-Tattoo” Glucose Sensors

0

2

4

6

8

10

12

14

16

18

20

0 5 10 15 20 25 30

Concentration of glucose (mM)

Per

cen

tag

e ch

ang

e in

FIT

C/T

RIT

C

rati

oy = 1.3116x - 0.0327

R2 = 0.9998

0

2

4

6

8

10

12

14

16

18

0 2 4 6 8 10 12 14


Per

cen

tag

e ch

ang

e in

FIT

C/T

RIT

C r

atio

0

2

4

6

8

10

12

14

16

18

20

0 5 10 15 20 25 30


Per

cen

tag

e ch

ang

e in

FIT

C/T

RIT

C

rati

oy = 1.3116x - 0.0327

R2 = 0.9998

0

2

4

6

8

10

12

14

16

18

0 2 4 6 8 10 12 14


Per

cen

tag

e ch

ang

e in

FIT

C/T

RIT

C r

atio

H2O, Small Molecules

Indicator Molecules

h out

h in

Fluorescent images of alginate microspheres before and

after citrate treatment (Dual filter, 488/ 550nm)

Continuous sensing profile and sensitivity curve of alginate microsphere glucose sensors under dynamic conditions

in DI water. Mean ± SD (n=5)


Status and Results

• Reliable microsphere and capsule fabrication technology

• Immobilization/encapsulation developed for many indicators, enzymes and RET assays

• Improved encapsulation using dissolved core microspheres

• Initial tox screening & in vivo biocompatibility

• Dynamic flow testing completed

• Instrumentation design and sensor response in vivo in progress

Title – Fluorescent Microsphere Implants

Chaudhary and Srivastava, Sensor Letters, 2008.


The Droplet Generator

Aerodynamically assisted jetting equipment

Nozzle diameter= 0.35mm

Product flow rate and pressure inside the chamber

http://www.nisco.ch/31.htm Size of the microspheres (50 - 80 μm)


Dissolution of alginate microspheres

LbL coated dissolved core

alginate mcirospheres

AF-647—dextran amino/

QSY-21-apo-GOx sensing

assay complex

Poly (sodium 4-styrene-sulfonate)

LbL coated algiante

microsphere containing

NIR sensing assay

LbL coated alginate

microspheres during

core dissolution

AF-750 labeled Poly-(allylamine hydrochloride)

Calcium ionsCrosslinking in alginate

microspheres

0.1M sodium

citrate-Tris HCl

0.1M sodium

citrate-Tris HCl

LbL coated dissolved core

alginate mcirospheres

AF-647—dextran amino/

QSY-21-apo-GOx sensing

assay complex

Poly (sodium 4-styrene-sulfonate)

LbL coated algiante


NIR sensing assay

LbL coated alginate

microspheres during

core dissolution



microspheres

0.1M sodium

citrate-Tris HCl

0.1M sodium

citrate-Tris HCl

LbL coated algiante


NIR sensing assay

LbL coated alginate

microspheres during

core dissolution



microspheres

0.1M sodium

citrate-Tris HCl

0.1M sodium

citrate-Tris HCl

Chaudhary et al, JDST 2010, Analyst, 2010, Botech Bioengg 2010.


Immunomodulating Agents

e.g. Dexamethasone or

NSAID’s e.g. Celecoxib or Naproxen

• More effective in action

• No Inflammation

• Minimally Invasive

• High patient Compliance

Glucose Sensor

Proposed Approach

for Smart Glucose Sensing

Anti inflammatory

agents

LBL

coating

Ref

Fluorophore

FRET

Sensing

Assay

Jayant, McShane and Srivastava, Drug Delivery 2009.


In vitro release studies:

2. Effect of different ratios of uncoated and (PAH/PSS)1 coated MS

Comparative release profile of uncoated and different combination of uncoated and

(PAH/PASS)1 coated dexamethasone loaded alginate microspheres in PBS (pH 7.4)

containing sodium azide (0.1%) at 37ºC, Mean± SD (n=3)

0

20

40

60

80

100

0 3 6 9 12 15 18 21 24 27 30

Time(d)

% D

ex R

ele

ase

25CP:75P 50CP:50P 75CP:25P Plain MS

Jayant and Srivastava, Drug Delivery 2009


In Vivo Studies

1 week

2 week

3 week

4 week

Figure. Pharmacodynamic changes in

representative subcutaneous tissue

sections of rats implanted with (I) Apo-

GOx alginate sensor, (II) Apo-GOx

sensor+(25P:75CP) dexamethasone

loaded alginate MS over (A) week 1; (B)

week 2; (C) week 3, and (D) week 4

post-implantation. Inflammation-

mediating cells and normal cells are

stained purple and pink, respectively

(H&E staining)

Jayant, McShane and Srivastava, IJP 2011


Product Concepts

• Biocompatible fluorescent microspheres or

microcapsules

– Implanted in dermis by surgeon or licensed

provider

– Optical readout system calibrated after initial

healing period

– Daily one-point calibration—if needed

• Alternatively, can be integrated with a ex-

vivo system for sensing various analytes

Title – Fluorescent Microsphere Implants


Portable Nanoenginereed Urea

Biosensor for Dialysate Monitoring

• Flow-through chamber with biosensor film on wall

– Slide changes color (absorbance) with pH

• Two LEDs for tracking color change (ratiometric)

• Photodiodes that detect the signals

Photodiode Flow chamber

Voltage

Output

Signal Generator

Signal Generator

LEDs


Placement of Device

Hemodialysis

Machine Urea

Biosensor Computer

Blood Flow to

dialysis

machine

Blood Flow

from dialysis

machine

Spent

Dialysate

Dialysate

Collection

Data

Transmission

Fresh

Dialysate


Optical Transduction

• Based on the observed colour change of a pH sensitive dye

• No reference required when using a ratiometric dye

• Not affected by electrical interferences

• Highly sensitive

• Simple and cost effective

NH2CONH2 + 3H2O →2NH4+ + HCO3

− + OH−


Status and Results

• pH-sensitive microspheres made & tested

• Urea sensing completed in standard urea

solutions

• Urea sensing completed in dialysate waste

fluid from KEM hospital

• Inexpensive monitoring instrument built &

tested

• Integration complete using a MEMS device

Title – Urea Biosensor for Dialysate Monitoring


Tygon

Tubing

Disposable Plastic Cuvette

Tygon

TubingSensing Film with

Dye & Enzyme

Bio-recognition element

Urease immobilized within calcium alginate microspheres

Transducer

Cresol red dye within nanofilms

Sensor Assembly

Swati and Srivastava, Analytical Letters, 2009.


0

0.2

0.4

0.6

0.8

1

1.2

0 2 4 6 8

urea concentration (mM)

Ab

so

rban

ce r

ati

o (

572/4

28)

0.035

0.085

0.135

0.01 0.06

Figure: Uncoated calcium alginate

microspheres

Figure: Coated alginate

microspheres

Figure: Urea sensing with microspheres: Minimum

detection limit of urea is 0.01 mM

Figure: Cresol red immobilized in

nanofilm coatings

Approach to sensor fabricationUrease/Cresol red encapsulated microspheres

0

0.2

0.4

0.6

0.8

1

1.2

6.5

6.7

6.9

7.1

7.3

7.5

7.7

7.9

8.1

2 4 6 8 10

Ab

so

rban

ce r

ati

o

pH

Time (minutes)

pH of buffer

pH of urea solution

Absorbance ratio with buffer

Absorbance ratio with urea solution

Figure: Response

of biosensor

Flow cell set up

Figure: Flow cell set up with white light

source, Spectrophotometer

(USB4000), Slide holder (Top image)

Magnified view of flow cell with tubings

( bottom image)

Swati and Srivastava, ACA, 2010.


Product Concepts

• Disposable tubing or tubing-connected cell

• Single optoelectronic instrument attached

directly to disposable tubing/cell

• Software/hardware interface to dialysis

system

Title – Urea Biosensor for Dialysate Monitoring


Nano-in-Micro Biosensors

Nanosensors

Wavelength (nm)Wavelength (nm)

Inte

nsit

y

Inte

nsit

y

Lactate sensor

h em (indicator)

h em (reference)

h ex

Glucose sensors etc

Multilayer Coatings

Microsphere carrier

Nanosystems

CLSM images of FITC–dex loaded

gelatin nanoparticles (1a), gelatin-in-

alginate microspheres (1b) and

CaCO3 nanoparticles (2a), and

CaCO3-in-alginate microspheres

(2b). Differential interference

contrast images of gelatin-in-alginate

microspheres (1c) and CaCO3-in-

alginate microspheres (2c).

Joshi and Srivastava, Carb Polym, 2010.


Multi-analyte sensor using MEMS device

Mechanism for glucose biosensing: The

H2O2, released during glucose oxidation by

Glucose oxidase enzyme (GOD), acts as an

electron acceptor, thus quenching the

fluorescence of QDs (CdSe-CdS).


• US patent ― Glucose biosensor system coupled with an anti-inflammatory module and methods for using the same‖, Date of filing - June 21, 2010, Application no.- 12/819,868

• Indian patent ―Glucose biosensor system coupled with an anti-inflammatory module and methods for using the same‖, Date of filing - April 23, 2010, Application no.- 1319/MUM/2010

• US patent ―BIOSENSOR FOR HEALTH MONITORING AND USES THEREOF‖, Date of filing – July 15, 2010, Application no.-12/837,218

• US patent ―-COMPOSITIONS AND METHODS FOR NANO-IN-MICRO PARTICLES‖, Date of filing – Mar 22, 2010, Application no.- 12/728,936

• Indian patent with Prof D Bahadur ―A Stable Delivery System for Statin Family Drugs‖, Application No - 2093/Mum/2008

• Indian patent with Prof D Bahadur and Prof M Aslam: “Multilayer Nanocomposite‖, Date of filing – 21st March 2011, Application No - 811/MUM/2011.

Patents Applied


Acknowledgments

• DBT for funding the glucose sensor project

• BRNS for funding the drug release project

• DST for funding the nanosensor project

• ICMR for funding the urea sensor project

• CSIR for funding the lactate sensor project

• DBT NM for funding the Photothermal therapy cancer project

• DST NanoMission for funding the Polymeric Nanoparticles project

• DBT IYBA for funding the Nano-in-Micro project

• BRNS-DAE for funding the NIR glucose sensor project

• DBT-Bioengg for funding the Calcium Sensor Project

• Students in NanoBios lab especially Ayesha, Rahul, Abhijeet and Swati for the biosensor work

• Colleagues at IIT Bombay and collaborators across the world!

nanoengineered biosensors presentation srivastava aug 24 2011 · 2011-11-17 · srivastava and...

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