dendrimers based electrochemical...

INTRODUCTION

Dendrimers are hyperbranched globular

macromolecules with well-defined, mono-

disperse, three dimensional spatial

conformations, and a wide spectrum of

chemical and physical properties (Tomalia et

al., 1985). These characteristics indicate

significant differences from the classical

polymeric molecules. Structurally, these

macromolecules are divided into three

architectural regions: the central core,

repetitive and radial branching units and the

terminal functional groups. To achieve a high

degree of precision and structural order,

dendrimers are synthesized in a stepwise

fashion. The number of repeat branching

molecules used during the synthesis refers to

the generation of dendrimers, which also

governs the shape and size of the dendrimers.

Generally, two different methods namely,

divergent and convergent, are adopted for the

Key words: Dendrimers, Biosensors, Polyamidoamine, Polypropylene imine, Bioreceptors, DNA sensor. *Corresponding Author: Dhirendra Bahadur, Department of Metallurgical Engineering and Material Science, Indian Institute of Technology Bombay, Powai, Mumbai, India. Email: [email protected]

Dendrimers based Electrochemical Biosensors

Electrochemical biosensors are portable devices that permit rapid detection and monitoring of biological,

chemical and toxic substances. In the electrochemical biosensors, the bioreceptor is incorporated into the

transducer surface; and when in contact with the analyte, generates measurable signals proportional to the

analyte concentration. Materials with high surface area, high reactivity, and easy dispersability, are most

suited for use in biosensors. Dendrimers are nanomaterial gaining importance for fabrication of

electrochemical biosensors. These are synthetic macromolecules with regularly branched tree-like and

globular structure. The potential applications of dendrimers as biosensors are explored due to their

geometric symmetrical structure, chemical stability, controlled shape and size, and varied surface

functionalities, with adequate functional groups for chemical fixation. The current review provides multi-

faceted use of dendrimers for developing effective, rapid, and versatile electrochemical sensors for

biomolecules. The redox centers in the dendrimers play an important role in the electron transfer process

during immobilization of biomolecules on the electrodes. This has led to an intensive use of dendrimer

based materials for fabrication of electrochemical sensors with improved analytical parameters. The review

emphasizes development of new methods and applications of electrochemical biosensors based on novel

nanomaterials.

1 2 1Saumya Nigam , Sudeshna Chandra , Dhirendra Bahadur *

1Department of Metallurgical Engineering and Material Science, Indian Institute of Technology Bombay, Powai,

Mumbai, India2Department of Chemical Sciences, School of Science, NMIMS (Deemed-to-be) University, Vile Parle (W), Mumbai,

India

Biomed Res J 2015;2(1):21-36

Review

synthesis of dendrimers, and classified into

different “generations”. It is the hyper-

branching of the molecule from the centre of

the dendrimer towards the periphery that

results in homostructural layers between the

focal points (branching points). The number of

focal points from the core towards the outer

surface is the generation number. Thus,

generation refers to the number of repeated

branching cycles performed during the

synthesis. The core part of the dendrimer is

denoted generation “zero” (G0). For example

if a dendrimer is made by convergent

synthesis, and the branching reactions are

performed onto the core molecule three times,

the resulting dendrimer is considered a third

generation dendrimer. Each successive

generation results in a dendrimer roughly

twice the molecular weight of the previous

generation.

The two synthetic methods have inherent

advantages and disadvantages. Using the

divergent synthesis method, the dendritic

molecule is formed from a central core which

then extends radially outwards through

addition of branching molecules. The main

advantage of the divergent method is that high

molecular nanoscaffold architecture is

attained with desired repetitive branching

monomers. Thus, the dendrimer can be tailor

made to achieve maximum functionalities and

properties. However, two major challenges are

encountered in divergent synthesis. First, the

number of reaction points increase in

geometric progression with every generation

followed by increase in molecular weight.

This compromises the reaction kinetics,

making it slower and synthesis of high

generation dendrimers becomes difficult,

further lowering the yield of desired product.

Addition of each branching unit requires care

and precision to prevent structural defects and

asymmetry in the dendrimer structure.

Secondly, the separation of desired dendrimer

from the by-products is hindered due to

molecular similarity exhibited by the by-

product as well as the desired dendrimer. On

the other hand, convergent method employs

synthesis of small dendrites from the exterior

and the reaction proceeds inwards to the

central core. The convergent procedure results

in lesser structural defects and easy

purification of dendrimers resulting in high

degree of monodispersity. Despite the

possibility of purer and flawless dendrimers,

the convergent method falls short in synthesis

of higher generation dendrimers. This choice

is limited due to the steric forces crowding the

dendrites around the central core molecule.

Despite the difficulties, these macro-

molecules have gained interest over classical

polymers due to the varied options presented

by dendritic macromolecules. The freedom of

choice of central core, branching monomeric

units and surface functional groups from the

vast pool of molecules gives rise to a

multivalent system. Ethylenediamine, 1,4-

diaminobutane, 1,12-diaminododecane,

Biomed Res J 2015;2(1):21-36

22 Dendrimers based electrochemical biosensors

cystamine, 1,6-diaminohexane and ammonia

are the most common core molecules. The

varied core and branching monomers affect

the internal chemical environment, three

dimensional structures and size of internal

cavities in the dendrimer. Due to the different

structural and chemical properties, these user-

customized dendrimers find applications in

the fields of drug delivery, gene delivery,

antimicrobials, magnetic resonance imaging,

immunosensing and biosensing.

Methyl acrylate alternating with ethylene

diamine forms the most widely synthesized,

studied and used class of polyamidoamine

(PAMAM) dendrimers (Esfand et al., 2002),

with the internal amide groups providing an

abundance of lone pairs of electrons. Another

popular class of amine terminated dendrimers

is the poly (propylene imine) (PPI)

synthesized by Michael's addition of primary

amines to acrylonitrile followed by

subsequent hydrogenation by Raney cobalt or

Raney nickel catalyst (de Brabander-van den

Berg et al., 2003). The interiors of PPI

dendrimers are the tertiary nitrogen atoms with

lone pairs of electron contributing to their

reactive cavities. Both the classes of

dendrimers have primary amine groups on the

surfaces governing the surface properties,

reactivity and surface charge. Thus, any kind

of detection response observed in these

dendrimers is attributed to the amine groups.

The surface of dendrimers is further modified

to enhance the reactivity/interaction and

Biomed Res J 2015;2(1):21-36

sensor response to be used in biosensing

applications. Various molecules like

ferrocene, polystyrene, polyaniline,

carbohydrates, etc. have been explored for

surface modification (Ashton et al., 1997;

Chen et al., 2014; Hung et al., 2013; Yoon et

al., 2000). The conductivity of the moieties

plays an important role in enhancing response

of the dendritic scaffold in sensing various

biomolecules. The most common modifying

molecule is ferrocene which exhibits multi-

electron transfer in various redox interactions.

Ferrocene has been exploited as central core,

branching monomer as well as for surface

groups in various dendritic systems (Mehmet

et al., 2013; Villalonga-Barber et al., 2013).

They behave as non-interacting redox

moieties undergoing redox processes without

decomposition while maintaining the desired

electrochemical reversibility (Sun et al.,

2014).

The molecular recognition of

biomolecules by dendrimers is primarily

governed by the three dimensional

conformation in higher generations. The

branches of lower generation dendrimers tend

to radiate out towards the periphery and exist

in open conformation. On the other hand, as

the number of generation is increased, the

branches tend to retract and adopt globular

conformations in a three dimensional space

with intramolecular hydrogen bonding

governing the structures. The generation

dependent conformational changes confirmed

Nigam et al. 23

by X-ray analysis, demonstrated that the

higher generations are more spherical as

compared to lower linear generations (Percec

et al., 1998). The globular conformations

closely resemble morphology of globular

proteins and are useful in several biosensing

applications associated with the biomimetic

macromolecular architecture. A vast variety of

biomolecular species have been detected using

dendrimer scaffolds. In the following sections,

details of the various sensors using different

types of dendrimers are discussed.

Dendrimers in electrochemical biosensing

By definition, a biosensor is an analytical

device that makes use of bioreceptor molecule

immobilized onto a transducer (recognition)

surface and produces measurable signals in the

presence of an analyte, due to the bio-

recognition event proportional to the

concentration of the analyte. Biosensors are

classified based on either the bioreceptor or

transduction method or both. Common

bioreceptors include enzymes, antibodies and

DNA, while transducers include electro-

chemical, piezoelectrical, optical techniques.

The transducer techniques using electro-

chemical biosensors have an edge over other

methods due to excellent selectivity and

sensitivity, and precise detection of the desired

species. These are relatively cheaper, faster

and more user friendly as compared to other

techniques. The exceptional features render

the electrochemical biosensors increasingly

applicable in several biomedical and

environmental analyses.

a) Peroxide sensor: Copolymers of pyrrole-

PAMAM dendrimer are used for

electrochemical sensing of hydrogen

peroxide. Different generations of

pyrrole-PAMAM with branched amine

periphery and focal pyrrole functionality

are synthesized by divergent method. The

conjugate is covalently attached to the

electrode surface and horseradish

peroxidase (HRP) immobilized on it to

form conducting films for H O sensing. 2 2

The steady state amperometric response is

measured as a function of H O 2 2

concentration at +0.35V vs. Ag/AgCl, and

demonstrated that the dendritic wedge

played an important role for

immobilization of the HRP enzyme

(Mehmet et al., 2012). Yang et al. (2014)

described a magnetic electrochemical

sensor comprising Fe O nanoparticles 3 4

with graphene oxide (GO) and subsequent

modification by PAMAM dendrimers.

The platform was employed for

modification of the gold electrode acting

as the working electrode and used for the

detection of H O in phosphate buffer 2 2

solution by the method of amperometric

i-t curve. The cyclic voltammograms of

Fe O /GO and Fe O /GO–PAMAM 3 4 3 4

showed an increase in current while

displaying steady redox peaks which

confirmed occurrence of a catalytic

Biomed Res J 2015;2(1):21-36


reaction on the electrode interface. H O 2 2

was detected in a linear calibration range –5 –3of 2.0 × 10 –1.0 × 10 M with a

correlation coefficient of 0.9950 and -6detection limit of 2.0 × 10 M. The sensor

platform also displayed excellent recovery

ratios of 96.9–108.1% H O added to milk 2 2

and juice samples. Another amperometric

electro-chemical biosensor for H O was 2 2

developed by modifying gold bead

electrodes with starburst PAMAM

dendrimers of different generations of 2, 3

and 4, followed by absorption of Prussian

blue (PB). The covalently bonded

dendrimer/PB modified electrodes offered

enhanced sensitivity and lower detection

limits (Bustos et al., 2006). Metallic

(Rhodium) nanoparticles stabilized with

N, N-bis-succinamide-based dendrimer

were immobilized on glassy carbon

electrode (GCE) and electrocatalytic

activity towards hydrogen peroxide

reduction investigated using cyclic

voltammetry and chronoamperometry.

The dendrimer stabilized nanoparticles

showed excellent electrocatalytic activity

for H O reduction reactions and a steady-2 2

state cathodic current response was

observed at −0.3 V (vs. SCE) in phosphate

buffer (pH 7.0). The electrochemical

sensor displayed a linear response to H O 2 2

concentrations ranging from 8 to 30 μM

with a detection limit and sensitivity of 5 −6 −1

μM and 0.031 × 10 A μM , respectively

Biomed Res J 2015;2(1):21-36

(Chandra et al., 2009).

b) Glucose Sensor: A dendritic wedge based

on pyrrole-PAMAM dendrimer was used

to immobilize glucose oxidase (GOx) for

the construction of an amperometric

glucose sensor (Mehmet and Cevdet,

2012). Nanobiocomposite based glucose

biosensor was prepared by electro-

polymerization of pyrrole containing

PAMAM encapsulated Pt nanoparticles

(Pt-PAMAM), and GOx. The developed –1sensor had a sensitivity of 164 µA mM

–1cm and a detection limit of 10 nM within

a wide working range from 0.2−600 µM.

Pyrrole provided electrical conductivity,

stability and homogeneity to the thin film,

while PAMAM provided a favorable

microenvironment to maintain bioactivity

of GOx (Tang et al., 2007). Yoon and

colleagues used varying degrees of redox-

active ferrocenyl in combination with

PAMAM dendrimers (Fc-D) as

recognition unit for fabrication of a

glucose sensor (Yoon et al., 2000). GOx

was deposited layer-by-layer on Au-

surface to form an enzymatically active

GOx/Fc-D multilayered assembly. The

bio-electrocatalytic signals from the

multilayer were directly correlated to the

number of layers deposited, confirming

the tunable sensitivity of the electrode and

hence a potential microbiosensor. Cyclic

voltammetry and surface plasmon

resonance (SPR) was used to investigate

Nigam et al. 25

the redox-orientation changes of

ferrocene-tethered dendrimers and GOx.

SPR monitors change in the refractive

index of the medium next to the Au

sensing surface and are used to monitor

immobilization of GOx onto the Au

surface (Frasconi et al., 2009). Redox-

active dendrimer fabricated using

different generations of poly (propylene

imine) core with peripheral octamethyl

ferrocenyl units (Fig. 1) and deposited on

Pt electrodes for immobilizing GOx has

been used for detection of glucose

(Armada et al., 2006). The amperometric

response of all the dendritic mediators

towards glucose was determined at several

applied potentials. Glucose biosensor has

been developed based on bioactive

polyglycerol (PGLD) and chitosan

dendrimer (CHD). Both the dendrimers

were conjugated with GOx to form

PGLD-GOx and CHD-GOx and

entrapped in polyaniline nanotubes

(PANINT's) during template electro-

chemical polymerization of aniline. The

prepared PGLD-GOx/PANINT's and

CHD-GOx/PANINT's biosensors

exhibited strong amperometric response

to glucose concentrations in ranges

observed in human blood. PGLD-

GOx/PANINT's was more sensitive –1(10.41 nA.mM ) as compared to CHD-

–1GOx/PANINT's (7.04 nA.mM ), due to

specific organization of the GOx layer at

the surface of PGLD and distribution of

PANINT's (Santos et al., 2010).

Ferrocenyl dendrimer (PAMAM-Fc)

has also used for fabricating an

amperometric glucose biosensor. Series of

asymmetric PAMAM dendrimers

containing a single ferrocene unit located

in the focal point have been synthesized.

The transducer consisted of a gold

electrode covalently modified with 3-

mercaptopropionic acid, PAMAM-Fc

dendrimers and GOx enzyme. The

PAMAM-Fc/GOx biosensor showed

excellent performance for recognizing

glucose at +0.25 V with a high sensitivity

(6.54 μA/mM) and low response time

(~3s) in the concentration range of 1–22

mM (Mehmet et al., 2013).

Figure 1: Structures of varying generations of octamethyl

ferrocenyl dendrimers for use as electrode material for

determination of glucose (Armada et al., 2006).

Biomed Res J 2015;2(1):21-36


c) DNA Sensor: Dendrimers were also

exploited for their possible use in

fabricating DNA sensors. An electro-

chemical DNA nanobiosensor was

developed by immobilization of 20-mer

thiolated probe ssDNA on electro-

deposited poly (propyleneimine)

dendrimer (PPI) of generation 4 (G4),

doped with gold nanoparticles (AuNP)

(Arotiba et al., 2008). Cyclic voltammetry

showed that the designed platform

(GCE/PPI-AuNP) exhibited reversible

electrochemical behavior in pH 7.2

phosphate buffer saline (PBS) solution

due to PPI. The redox chemistry of PPI

involves a two electron and one proton

process and is pH-dependent. PPI-AuNP

was able to amperometrically detect target

DNA concentrations at 0.05 nM in PBS.

Using electrochemical impedance

spectroscopy (EIS), the biosensor –12 –9exhibited a dynamic linearity of 10 –10

M for target DNA. The probe immobiliza-

tion effectiveness is apparently attributed

to the AuNP's ability to connect to the

thiolatedssDNA on the GCE surface via

Au-S linkages. Further, the electrostatic

interaction between the cationic platform

and the anionic DNA probe improved the

immobilization process. Proposed charge

transfer scheme between the electrolyte,

DNA and PPI-AuNP is shown in Fig. 2.

A DNA biosensor with probe DNA

sequence, immobilized on a multinuclear

Biomed Res J 2015;2(1):21-36

nickel (II) salicylaldimine metallo-

dendrimer on GCE has been reported

(Arotiba et al., 2007). The authors studied

electrochemical characterization on

immobilization layer of the PPI derivative

by impedimetric and amperometric

methods. The metallo-dendrimer was

electroactive with two reversible redox

centers and was a strong DNA adsorbant.

The sensor responded to 10 µL of 5 nM

target DNA with detection limit as low as –12 3.4 × 10 M. Gold electrode has been

modified with 3-mercaptopropionic acid

and reacted with amino-terminated

PAMAM G-4 dendrimer to obtain a thin

film (Li et al., 2009). Recognition layer of

single-stranded 3´-biotin-avidin combina-

tion was immobilized onto the thin film to

detect the complimentary target. Cyclic

voltammetry (CV), differential pulse

voltammetry (DPV) and electrochemical

impedance spectroscopy (EIS) has been

used to study immobilization and

hybridization of DNA. The dynamic

detection range of the sequence-specific –11 –14DNA was 1.4 × 10 –2.7×10 M with a

–14detection limit of 1.4 × 10 M. Sahoo et

al. (2013), demonstrated a label free

Figure 2: Proposed charge transfer scheme between

PBS, DNA and PPI-AuNP (Arotiba et al., 2008).

Nigam et al. 27

impedimetric DNA biosensor based on

third generation G3 PAMAM dendrimer

functionalized GaN nanowires (NWs).

The developed nanosystem provided large

docking sites to immobilize probe (p-)

DNA covalently. The biosensor was

ultrasensitive and showed detection limit

as low as attomolar (aM) concentration of

complementary target (t-) DNA.

Impedance spectroscopy revealed an

increase in the resistance polarization (R ) p

indicating efficient charge transfer due to

strong covalent binding on NWs surface.

Zhu et al. (2006) modified gold electrodes

with sub-monolayers of mercaptoacetic

acid (RSH) and reacted with G-4 PAMAM

dendrimers to obtain thin films of

PAMAM/RSH. DNA probe was then

immobilized onto the thin films to afford

stable recognition layers. DPV was used to

monitor DNA hybridization with

daunomycin (DNR) as indicator. The

PAMAM-modified Au electrodes without

ssDNA showed good electrochemical

response in DNR solution, while on

attachment with ssDNA the modified

electrode showed a decrease in the DPV

response of DNR. This is attributed to less

accessibility of DNR molecules to ssDNA

probe on the electrode surface. Besides

high generation dendrimers, low

generation dendrimers are also used to

develop DNA biosensor. A second

generation PAMAM (G2-PAMAM)

dendrimer was covalently functionalized

onto multi-walled carbon nanotube

(MWNT) and used as electronic

transducer and tether for surface

confinement of probe DNA. Impedance

spectroscopy revealed occurrence of

hybridization between surface confined

ssDNA probe with target DNA in solution

to form double stranded DNA (dsDNA).

The interfacial charge-transfer resistance

of the electrode towards the redox

electrolyte changed due to occurrence of

hybridization. The large number of amino

groups of the dendrimer enhanced the

surface binding of the probe DNA which

in turn resulted in increase in the

sensitivity of the impedimetric biosensor

for the target DNA. The interfacial charge-

transfer resistance responded linearly to

the logarithmic concentration of the target

DNA within a concentration range of

0.5–500 pM with a detection limit of 0.1

pM (S/N = 3) (Zhu et al., 2010). Single-

use electrochemical DNA biosensor has

been fabricated based on pencil graphite

electrode modified with succinamic acid

and G2 PAMAM dendrimer (G2-

PS/GCE). Calf thymus double stranded

DNA (ctDNA) and DNA oligonucleotide

(DNA ODN) immobilized on surface of

G2-PS/GCE under optimum conditions,

showed a detection limit of 4.2 µg/mL

(Congur et al., 2014). Besides dendrimers,

dendritic nanostructures have been used

Biomed Res J 2015;2(1):21-36


dendrimer. Thrombin aptamer probe was

immobilized onto activated dendrimer

monolayer film and detection of thrombin

was investigated in the presence of the 3−/4−reversible [Fe(CN) ] redox couple 6

using impedance technique. The results

showed that the charge-transfer resistance

(R ) value had a linear relationship within ct

concentrations range of 1–50 nM

thrombin, and detection limit (S/N = 3) of

0.01 nM (Zhang et al., 2009).

Impedimetric aptasensor based on

succinamic acid-terminated PAMAM

dendrimer was developed for monitoring

interaction between DNA aptamer (DNA-

APT) and its cognate protein, human

as electrode material in biosensing

applications. Li et al. (2011) described

dendrimer-gold (Den-Au) nanostructure

modified electrode by directly placing the

electrode into 2.8 mM HAuCl and 0.1 M 4

H SO solution at –1.5 V. Scanning 2 4

electron microscopic images show growth

evolution of Den-Au at different time

period (Fig. 3). The Den-Au modified

electrode respond to 1 fM complimentary

target DNA within a wide detection range.

Aptamers, as single-stranded DNA or

RNA sequences that bind to specific target

molecules was determined by a label-free

highly sensitive impedimetric aptasensor

based on amino-terminated PAMAM

Figure 3: SEM images of Den-Au electrodes by electrodeposition in 2.8 mM HAuCl and 0.1 M H SO at different time 4 2 4

points (A) 20s, (B) 100s, (C) 300s and (D) 600s (Li et al. 2011).

Biomed Res J 2015;2(1):21-36

Nigam et al. 29

activated protein C (APC), a key enzyme

in the protein C pathway. The dendrimer

modified aptasensor showed detection

limits of 1.81 µg/mL in buffer solution and

0.02 µg/mL in diluted FBS (Erdem et al.,

2014).

d) Coenzyme Sensor: Hyperbranched

carbosilane polymers, polydiallyl methyl

silane (PDAMS) and polymethyl

diundecenyl silane (PMDUS) with

ferrocene moieties were used for

stabilization of Pt nanoparticles and as

electrode material for NADH oxidation.

The modified electrodes worked in wide

linear concentration ranges for NADH

with a detection limit of 4.78 µM for

PDAMS/PtNPs/Pt and 6.18 µM for

PMDUS/PtNPs/Pt. With regard to the

structure of the two carbosilane polymers

and their films, PDAMS with shorter

branches form rougher films and exhibit

higher rate constants (K ) and sensitivity obs

and smaller Michaelis constants (K' ), M

than PMDUS indicating better

electrocatalytic activity towards NADH

oxidation (Jiménez et al., 2014).

e) Other biomolecules: Tang et al. (2007)

reported enzyme based amperometric

biosensor for determination of glutamate.

A self-assembly of glutamate dehydro-

genase (GLDH) and PAMAM dendrimer

encapsulated Pt nanoparticles on carbon

nanotubes (GLDH/Pt-PAMAM) /CNT) n

were used as electroactive material (Fig.

4). The electrochemical activity was

reported to be attractive with large

determination range of glutamate (2–250

µM), short response time (< 3 s), high –1 2sensitivity (433 µA/mM cm ) and

stability.

Figure 4: Schematic showing the procedure of immobilizing Pt-PAMAM onto CNTs (a) layer-by-layer self-assembly of

GLDH and Pt-PAMAM onto CNTs (b) Pt-PAMAM/CNTs heterostructures were covalently attached via EDC (Tang et al.

2007).

Biomed Res J 2015;2(1):21-36


Pt-PAMAM and GLDH were

alternately deposited until suitable layers

were obtained. PAMAM G-4 dendrimers

crosslinked with reduced graphene oxide

were tested for performance as electro-

chemical biosensors by immobilizing

enzyme tyrosinase (Araque et al., 2013).

The bioelectrode showed excellent

electrocatalytic behavior towards

determination of catechol with a response

time of about 6s, linear range of 10 nM to –122 µM, sensitivity of 424 mAM and a

low detection limit of 6 nM (Fig. 5).

PAMAM dendrimer encapsulated

AuNPs were first immobilized to a

conducting polymer with two amine

groups (3',4'-diamine-2,2',5',2''-terthio-

phene (PDATT) through covalent bonding

between –COOH group of PAMAM and

–NH group of PDATT. Laccase was 2

subsequently covalently bonded to the

–COOH of PAMAM dendrimers to form

PDATT/Den (AuNPs)/laccase probe

(Rahman et al., 2008). The modified

electrode displayed direct electron-

transfer (DET) process of laccase and a

catechin biosensor was fabricated based

on the electrocatalytic process of laccase.

The linear range and detection limit for

catechin sensing was 0.1–10 and 0.05 µM,

respectively. An electrochemical

biosensor based on PAMAM dendrimers

was developed for the detection of

fructose in food samples by immobilizing

fructose dehydrogenase (FDH) on

cysteamine and PAMAM dendrimers. The

concentration range of the enzymatic

biosensor was 0.25–5.0 mM fructose

(Damar et al., 2011). PAMAM dendrimers

were also used to enhance signal response

of a nanobiocomposite fabricated to

obtain an immunosensor for alpha-feto

protein (AFP) in human serum (Giannetto

et al., 2011). The binding of the dendrimer

with biologically active molecules like

antibodies can improve the activity and

Figure 5: (A) Amperometric response obtained with Tyr/PAMAM-Sil-rGO/GCE for different catechol concentrations at

E = –150 mV (B) FE-SEM image of Tyr/PAMAM-Sil-rGO (Araqueet al., 2013).app

Biomed Res J 2015;2(1):21-36

Nigam et al. 31

Biomed Res J 2015;2(1):21-36

sensitivity of the system. Response range

and precision were evaluated using cyclic

voltammetry (CV) and double step

chronoamperometry (DSCA) with limit of

detection of 3 ng/mL and limit of

quantification of 15 ng/mL. The enhanced

immunosensor could be useful for

monitoring prognosis of pregnancy and

occurrence of neoplastic diseases.

Recently, a redox-active silver-PAMAM

dendrimer nanostructure was synthesized

in situ by using wet chemistry (Xiaomei et

al., 2013), and functionalized with mono-

clonal mouse anti-human antibody for free

prostate specific antigen (fPSA). Using,

graphite as the working electrode, a layer

of gold nanoparticles modified with

prostate-specific antibody (mAb2). In

presence of the fPSA, specific immuno-

complex was formed on the functionalized

antibody modified electrode. The Ag-

mediated PAMAM dendrimer directly

catalyzed reduction of H O in the 2 2

detection solution. Thus, PSA was

detected primarily due to the antigen-

antibody immunocoupling. Under optimal

conditions, the developed immunoassay

could determine target fPSA in the

dynamic range of 0.005–5.0 ng/mL with a

detection limit (LOD) of 1.0 pg/mL (S/N =

3). In addition, the accuracy of the

electrochemical immunoassay evaluated

for detection of clinical serum specimens,

was in accordance with referenced

enzyme-linked immunosorbent assay

(ELISA) method.

A multi-analyte sensing device based

on PAMAM dendrimer for simultaneous

at-line monitoring of glucose, ethanol,

pO - and cell density was fabricated (Akin 2

et al., 2011). The device consisted of a

dual biosensor, a modified microscope

and a fiber optical pO -sensor integrated 2

into a flow analysis (FA) system. The

electrochemical transducer consisted of

self-assembly of cysteamine on gold

surface. Alcohol oxidase and pyranose

oxidase were immobilized onto the gold

surface by means of PAMAM (poly-

amidoamine) dendrimer via glutar-

aldehyde cross-linking. The responses for

glucose and ethanol were linear up to 0.5

mM. The biosensor was used for

simultaneous determination of ethanol

and glucose in yeast fermentation process.

A highly stable and sensitive ampero-

metric biosensor was developed by

immobilizing alcohol oxidase (AOX)

through PAMAM dendrimers on a

cysteamine-modified gold electrode

surface for determination of ethanol (Akin

et al., 2009). The optimized ethanol

biosensor showed a linearity from

0.025–1.0 mM with 100 s response time

and detection limit (LOD) of 0.016 mM.

The analytical characteristics of the

system were also evaluated for alcohol

determination in flow injection analysis


(FIA) mode for analysis of ethanol in

various alcoholic beverage as well as

offline monitoring of alcohol production

through yeast cultivation (Yuksel et al.,

2012).

PAMAM dendrimer (generation G4)

stabilized with 1-hexadecanethiol was

used for immobilization of acetylcholin

esterase from electric eel, and choline

oxidase from Alcaligenes sp. was used as

electrode material for fabrication of an

amperometric sensor for pesticides

(Snejdarkova et al., 2004). On similar

lines, urea electrochemical biosensor was

developed based on an electro-co-

deposited zirconia-PPI dendrimer

modified screen printed carbon electrode.

Urease enzyme was immobilized onto

electrodes and an amperometric response

in urea concentration from 0.01 mM to

2.99 mM was obtained with sensitivity of –1 –23.89 µA mM cm (Shukla et al., 2014).

PPI dendrimers have also been used to

reduce HAuCl to form core-shell PPI-Au 4

nanoclusters with several PPI molecules

attached on the surface of one gold

nanoparticles (Zhang et al., 2007). PPI-Au

nanoclusters and myoglobin (Mb) were

alternately adsorbed on the surface of

pyrolytic graphite (PG) electrodes

forming {PPI-Au/Mb} layer-by-layer n

films. The multilayer film assembled with

the dendrimer stabilized Au nanoparticles,

provided a new approach to fabricate

biosensors and bioreactors based on direct

electrochemistry of proteins and enzymes.

CONCLUSIONS

Contemporary studies indicate that the most

elementary chemical reaction of electron

transfer is widely prevalent in several

biological systems and more importantly in

nanosystems with redox dendrimers. This is

possible by tailoring the nature and topology

of the dendrimers to precisely control location

of the redox sites within the macromolecule

and study its electron-transfer processes. The

increase in efforts to combine dendrimers with

other molecules like pyrrole, ferrocene,

enzymes, etc. is promising in biosensing

applications.

ACKNOWLEDGEMENTS

The authors acknowledge Department of

Science and Technology, Government of

India, New Delhi, for providing financial

support. The authors also acknowledge the

publishers for providing copyright

permissions for the figures.

CONFLICT OF INTEREST

The authors claim no conflict of interest.

Biomed Res J 2015;2(1):21-36

Nigam et al. 33

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