Accepted Manuscript

Title: Electrochemical nanobiosensing in unprocessed whole blood: recent

advances

Author: Mohammad Hasanzadeh, Nasrin Shadjou

PII: S0165-9936(15)30031-5

DOI: http://dx.doi.org/doi: 10.1016/j.trac.2015.07.018

Reference: TRAC 14591

To appear in: Trends in Analytical Chemistry

Please cite this article as: Mohammad Hasanzadeh, Nasrin Shadjou, Electrochemical

nanobiosensing in unprocessed whole blood: recent advances, Trends in Analytical Chemistry

(2015), http://dx.doi.org/doi: 10.1016/j.trac.2015.07.018.

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1

Electrochemical nanobiosensing in unprocessed

whole blood: Recent advances

Mohammad Hasanzadeh a*

, Nasrin Shadjou b,c**

a Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz 51664,

Iran.

b Department of Nanochemistry, Nano Technology Center, Urmia University, Urmia,

Iran.

c Department of Chemistry, Faculty of Chemistry, Urmia University, Urmia, Iran

E-mail address:

(* ( Mhmmd_hasanzadeh@yahoo.com, hasanzadehm@tbzmed.ac.ir

(** ( Nasrin.Shadjou@gmail.com, N.Shadjou@urmia.ac.ir

Tel.: +98 914 3619877; fax: +98 41133632312.

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Highlights

Surfaces modification of nanomaterials for (bio/immune)-sensing applications in

whole blood was discussed.

Current developments in the determination of some biological molecules in

unprocessed blood were discussed.

Incorporation of biorecognition elements into nanomaterial based electrodes for

(bio/immune)-sensing in unprocessed whole blood was described.

Graphical Abstract

Abstract

Nowadays, the assay of whole blood is becoming increasingly important in modern

biomedical research such as clinical diagnostics, drug discovery, and biodefense. Whole

blood is a particularly complex mixture which contains a variety of substance such as

protein, glucose, inorganic salt, hormone, biomarkers and so on. Hence, a plentiful of

information can be obtained by investigating the whole blood, which is very helpful for

assessing the health status of the patient. Recently significant progress has been made in

electrochemical (bio/immune) sensing from whole blood using nanomaterials. This

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review describes fabrication and chemical modification of the surfaces of nanomaterials

for (bio/immune)-sensing applications in whole blood. We also present a comprehensive

overview of current developments and key issues in the determination of some biological

molecules with particular emphasis on evaluating the methods. We also discuss

incorporation of biorecognition elements into nanomaterial based electrodes for

immunesensing in unprocessed whole blood.

Keywords: electrochemical biosensing, nanotechnology, biomedical analysis, cancer,

enzyme, lab-on-a-chip, unprocessed and whole blood, immunesensing.

1. Introduction

Blood is one of the most informative sources for health and disease monitoring in the

human body [1] For example, monitoring levels of biomarkers in blood is known to be an

effective method for early diagnosis of various diseases such as cancer, by which better

treatment options and improved survival rates of patients can be provided [2].

Furthermore, therapeutic efficacy monitoring was demonstrated by following the levels

of chosen biomarkers in blood before and after a therapy, which facilitates the physicians

ability to determine the best treatment options [3]. Frequent monitoring of appropriate

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biomarkers is desirable for such purposes, since it leads to fast and timely feedback. This

approach requires an easily accessible sensory platform that can monitor the level of

biomarkers in a time-efficient and noninvasive (or negligibly invasive) manner with

comparatively low cost and minimal pain for the patients. However, few existing systems

satisfy all the aforementioned criteria. Thus, further research efforts are required toward

realization of such a system.

Nanobiosensors have the potential to meet the aforementioned criteria because of the

capability of performing rapid, label free, electrical detection with potentially low cost.

These devices utilize a capture agent on the sensor surface to bind the target biomolecules

with both selectivity and specificity. The captured biomolecules affect the electronic

properties of the nanowires, resulting in an electronically readable signal. Multiplexing

has also been demonstrated by selectively functionalizing the nanowires with receptors

for different biomarkers [4]. However, a challenge still remains toward making this

technology more clinically practical. That is, the use of whole blood as an input is not

typically investigated for biomarker detection; as such complex environments are known

to cause problems such as false signal and saturation of receptors. The use of whole blood

will allow evaluation of fragile proteins that experience prompt degradation after being

taken out of the body as well as providing a simplified sample preparation protocol to

expedite analysis.

Nowadays, the assay of whole blood is becoming increasingly important in modern

biomedical research such as clinical diagnostics, drug discovery, and biodefense. Whole

blood is a particularly complex mixture which contains a variety of substance such as

protein, glucose, inorganic salt, hormone, and so on. Hence, a plentiful of information

can be obtained by investigating the whole blood, which is very helpful for assessing the

health status of the patient. For example, glucose levels in blood can be related to the

diagnosis of diabetes, alcohol consumption, obesity and high cholesterol, while the

lactate concentration in blood is a biochemical indicator of anaerobic metabolism in

patients with circulatory failure. The determination of immunogenic tumor-associated

antigens level is very beneficial to clinical tumor diagnoses. The availability of a rapid,

simple, low cost, in situ whole blood assay with the capacity to detect a variety of

selected analytes would greatly benefit point-of-care or public health applications.

Recently significant progress has been made in biosensing from whole blood using a

capture release microfluidic chip [5] or from desalted serum; however, more effort is

needed for developing accurate and cost-effective systems that allow direct use of whole

blood samples prepared using simple tools such as finger pricks. In addition, there is still

a lack of understanding about biosensing using nanomaterials in complex media such as

serum and plasma. Therefore, this review describes fabrication and chemical

modification of the surfaces of nanomaterials for (bio/immune)-sensing applications. We

also present a comprehensive overview of current developments and key issues in the

determination of some biological molecules with particular emphasis on evaluating the

methods. This review shows how nanomaterials have made significant contributions in

the developments of electrochemical nanobiosensors, including immuno-, enzyme, DNA,

aptamer ones. More importantly, different aspects of the electrochemical biosensors such

as type of nanomaterials, detection techniques, analytes and the corresponding sensitivity

and sample matrix, as well as several noticeably prominent characteristics have been

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discussed in detail. Accordingly, research opportunities and future development trends in

these areas are discussed.

2. Type of application

2.1. Electrochemical enzyme nanobiosensing in unprocessed whole blood

Recently, an impressive number of inventive designs for enzyme-based electrochemical

sensing appeared. These types of sensor combine enzyme layers with electrochemical

transducers to produce a biosensor and promise to provide a simple, accurate and

inexpensive platform for patient diagnosis. Electrochemical methods are well suited to

enzyme investigation. Because electrochemical reactions give an electronic signal

directly, there is no need for expensive signal-transduction equipment. Moreover,

because immobilized probe enzyme can be readily confined to a variety of electrode

substrates, detection can be accomplished with an inexpensive electrochemical analyzer.

Indeed, portable systems for clinical testing and on-site environmental monitoring are

being developed. Sensitive electrochemical-signaling strategies based on the direct or

catalyzed oxidation of enzyme bases, and the redox reactions of reporter molecules or

enzymes recruited to the electrode surface by specific enzyme probe-target interactions

and charge-transport reactions mediated by the p-stacked base pairs have all been

demonstrated.

In this sub-section of review, we selectively review recent advances of electrochemical

enzyme nanobiosensing in unprocessed whole blood. Below, we include examples, and

compare and contrast enzyme biosensors based on nanomaterials. We also discuss the

feasibility of nanomaterials usage in enzyme biosensing, as these technologies might be

implemented to produce sensitive multiplexed assays for clinical diagnostics of diseases.

Until now, the primary methods of detecting blood glucose concentration are performed

by biochemical analyzers and glucose meters [6]. In hospital, the quantification of the

concentration of glucose is mainly carried out in serum samples by biochemical

analyzers, which are isolated from whole blood by centrifugation, but not in untreated

whole blood. The test results are influenced by the different model numbers of test

instruments and detection reagents, the treatment processes of blood samples, especially

additional centrifuging and too long measuring time from collecting a specimen of blood

to examination. As for commercial glucose meters, accurate results for glucose

concentration cannot be provided by a commercial glucose meter, due to the fact that

some defects cannot be ignored during its operation. For example, blood samples are

obtained from the fingertip peripheral but not veins, and doped easily with tissue fluid. So

the development of novel glucose biosensors for antifouling, rapid, highly sensitive, and

selective detection is of paramount importance for blood glucose concentration

monitoring in whole blood samples.

While an electrochemical biosensor directly used in whole blood, the biofouling of

electrode surface can be developed by platelet, fibrin and blood cell adhesion in the

complex environment of whole blood media. The biofouling of electrode surface will

bring catastrophic damage to the electron transfer between enzyme and electrode redox

center. As we know, anti-biofouling surfaces lie at the heart of several contemporary and

advanced technologies, ranging from coatings for biomedical implants, ship hulls, to

carriers for targeted drug delivery [7,8]. Hydrophilic surfaces of polymers like

polyethyleneglycol (PEG), with low values of polymer-water interfacial energy, show

resistance to protein adsorption and cell adhesion [9]. In this case, the hydrophilic

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polymer coating, carboxymethyl-PEG-carboxymethyl (CM-PEG-CM), was designed and

explored as antibiofouling surface for preparing for electrochemical glucose biosensor

that can be used directly for whole blood samples [10]. The biosensor was applied in

whole blood directly, which was based on the low values of polymer-water interfacial

energy, resistance to protein adsorption and cell adhesion. The entrapped GOx could

preserve its bioactivity and exhibited an excellent electrochemical behavior with a formal

potential of -0.29 V in phosphate buffer solution (PBS) (pH = 7.4). The results indicated

that the modified electrode can be used to determine glucose without interference from l-

ascorbic acid (AA) and uric acid (UA) with the low detection limit of 12.4 µM. The data

obtained from the biosensor showed good agreement with those from a biochemical

analyzer in hospital. The GOx biosensor modified with CM-PEG-CM will have essential

meaning and practical application in future that attributed to the effect of anti-biofouling

and good performance.

At the similar report by this research group, a novel electrochemical biosensor, which can

be evaluate the level of blood glucose with the help of antibiofouling technology, was

prepared by Sun and coworkers [11]. In this report, glucose oxidase (GOx) was

immobilizing on polyurethane-Pluronic F127 (PUF127) nanospheres modified glass

carbon electrode (GCE). Then, the electrochemical behavior of the biosensor in whole

blood was studied. The results of this report, indicated that GOx immobilized on the PU-

F127 nanospheres exhibited direct electron transfer reaction, which led to stable

amperometric biosensing for glucose with a detection limit of 11.4 µM in whole blood.

Interestingly, prposed sensor by this research group also offered suitable anti-interference

ability to ascorbic acid (AA) and uric acid (UA), especially when a detection potential of

-0.49 V was employed.

Also, small sample volume and in situ detection of glucose in human whole blood was

developed by using a screen-printed carbon electrode (SPCE) coupled with a paper disk

[12]. Interestingly, the SPCE was modified with graphene/polyaniline/Au

nanoparticles/glucose oxidase(Gr/PANI/AuNPs/GOD) composite and then covered by a

paper disk impregnated with the sample. After introducing PBS on the paper disk, the

electrochemical measurement was carriedout. The assay was based on measuring the

current decrease of flavin adenine dinucleotide (FAD) in GOD provoked by the enzyme-

substrate reaction using differential pulse voltammetry (DPV). It seem that, this new

paper-based electrochemical glucose sensor shows promise in applying point-of-care

(POC) device in whole blood tests, and particularly being appropriate for use in the

developing world and in resource-limited settings.

Interestingly, Picher et al [13] developed a lab-on-a-chip containing embedded

amperometric sensors in four microreactors that can be addressed individually and that

are coated with crystalline surface protein monolayers to provide a continuous, stable,

detection of blood glucose. It is envisioned that the microfluidic device will be used in a

feedback loop mechanism to assess natural variations in blood glucose levels during

hemodialysis to allow the individual adjustment of glucose. Reliable and accurate

detection of blood glucose is accomplished by simultaneously performing (a) blood

glucose measurements, (b) autocalibration routines, (c) mediator-interferences detection,

and (d) background subtractions. The electrochemical detection of blood glucose

variations in the absence of electrode fouling events is performed by integrating

crystalline surface layer proteins (S-layer) that function as an efficient antifouling

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coating, a highly-oriented immobilization matrix for biomolecules and an effective

molecular sieve with pore sizes of 4 to 5 nm. Picher and coworkers demonstrate that the

S-layer protein SbpA (from Lysinibacillus sphaericus CCM 2177) readily forms

monomolecular lattice structures at the various microchip surfaces (e.g. glass, PDMS,

platinum and gold) within 60 min, eliminating unspecific adsorption events in the

presence of human serum albumin, human plasma and freshly-drawn blood samples. The

highly isoporous SbpA-coating allows undisturbed diffusion of the mediator between the

electrode surface, thus enabling bioelectrochemical measurements of glucose

concentrations between 500 mM to 50 mM (calibration slope dI/dc of 8.7 nA/mM). Final

proof-of-concept implementing the four microfluidic microreactor design is demonstrated

using freshly drawn blood.

To work with whole blood samples, the process of separating plasma or serum is a

frequently required. Centrifugation is the most common method for this separation [14].

As an alternative, several research groups attempted to directly separate plasma from

whole blood using µ-PADs. Two general approaches for blood separation on µ-PADs

have appeared. One approach is to use heamagglutination between immobilized

antibodies and red blood cell antigen. The resulting plasma can be separated and flows

through the hydrophilic paper [15]. The other approach uses different types of paper, e.g.

blood separation membranes, connected with Whatman No.1 in either vertical [16] or

lateral platforms [17]. Currently, paper based microfluidics for blood separation has been

used for determining glucose [15], liver function [16] and, total protein [17] primarily

using colorimetric detection. However, analysis of biological markers from whole blood

sample based on ePADs few report has been reported.

For example, Songjaroen and coworkers [18] fabricated a new µ-PAD platform for

electrochemical detection of glucose from whole blood samples following plasma

isolation. Effective separation of the blood cells from the plasma fraction is essential for

reliable analysis of glucose. First, glucose levels are reduced when plasma specimens

remain in prolonged contact with red blood cells [19]. In addition, hemoglobin interferes

with the glucose assay [20]. Here, the wax dipping technique [21] was used to create the

microfluidic patterns on the paper because it allows for two different types of paper to be

joined together. The final device was composed of two blood separation zones that

directed isolated plasma to a middle detection zone. The resulting flow generated a

uniform gathering of plasma at the electrode and much better reproducibility of

electrochemical signals. The proposed ePADs work with whole blood samples with 24-

60% hematocrit without dilution, and the plasma was completely separated within 4 min.

Glucose in isolated plasma separated was detected using glucose oxidase immobilized on

the middle of the paper device. The hydrogen peroxide generated from the reaction

between glucose and the enzyme pass through to a Prussian blue modified screen printed

electrode (PB-SPEs). The currents measured using chronoamperometry at the optimal

detection potential for H2O2 (-0.1 V versus Ag/AgCl reference electrode) were

proportional to glucose concentrations in the whole blood. The linear range for glucose

assay was in the range 0-33.1 mM. The coefficients of variation (CVs) of currents were

6.5%, 9.0% and 8.0% when assay whole blood sample containing glucose concentration

at 3.4, 6.3, and 15.6 mM, respectively. Because each sample displayed intra-individual

variation of electrochemical signal, glucose assay in whole blood samples were measured

using the standard addition method. Results demonstrate that the ePAD glucose assay

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was not significantly different from the spectrophotometric method (p = 0.376, paired

sample t-test, n = 10).

Acknowledging the benefits of hyperbranched polymers and their nanoparticles, herein

china researchers [22] reported the design and synthesis of sulfonic acid group

functionalized hydroxyl-terminated hyperbranched polyester (H3O-SO3H) nanoparticles

and their biomedical application. In this report, a glucose biosensor was fabricated by

immobilizing the positively charged Au nanoparticles, H3O-SO3H nanoparticles and

glucose oxidase (GOx) onto the surface of glassy carbon electrode (GCE) [22]. It can be

applied in whole blood directly, which was based on the good hemocompatibility and

antibiofouling property of H30-SO3H nanoparticles. The biosensor had good

electrocatalytic activity toward glucose with a wide linear range (0.2-20 mM), a low

detection limit 12 µM in whole blood and good antiinterference property. The

development of materials science will offer a novel platform for application to substance

detection in whole blood.

To complete our discussion in this sub-section, Table 1 summarizes recent reports on

analytical parameters of some electrochemical enzyme nanobiosensing methods in

unprocessed whole blood. In conclusions of this section, it is important to point out;

nanomaterials have emerged as versatile tools for generating excellent supports for

enzyme stabilization due to their small size and large surface area. By proper surface

modification, various nanomaterials have been synthesized and successfully utilized for

protein/enzyme immobilization, which have already displayed promising effects in

practical applications. We have summarized in this sub-section the applications of

nanomaterials in enzyme immobilization and bioanalysis. The immobilized enzymes

generally show better stability towards pH and heat than the free ones and can be

recovered and reused multiple times. However, the activity of some enzymes decreases to

some extent after immobilization, which indicates that more efforts are still required to

explore the immobilization techniques

On the other hand, up to now; almost all researches on nanomaterials based enzymatic

biosensors are still in laboratory. As for practical applications, few enzymatic biosensors

appear to be commercially feasible except for some blood glucose biosensors. As for

practical applications, few enzymatic biosensors appear to be commercially feasible

except for some blood glucose and hand-held biosensors. To commercialize the

enzymatic biosensors based on nanomaterials, efforts should be made to break some key

technical barriers such as controlling the morphology of nanomaterials on device,

realizing efficient enzyme immobilization and keeping enzyme long-life bioactivity as

well as reducing matrix interference and sensor fouling.

2.2. Electrochemical nano-immunesensing in unprocessed whole blood

Recently, there was growing interest in the use of electrochemical immunosensors for

clinical diagnosis [23]. Electrochemical immunosensors utilize various types of electrode

[e.g., as screen-printed carbon electrodes (SPCEs), carbon-paste electrodes (CPEs) and

glassy-carbon electrodes (GCEs)]. The antibodies were immobilized by physical

adsorption or chemical immobilization. These types of electrodes can be used only once.

With the selected latest research articles from 2012 to May 2015, herein, we summarize

various electrochemical nano-immunosensors for detection of different biomarkers in this

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review. More importantly, we discuss in detail different aspects such as type of

nanomaterials, injection and detection techniques, labels, analytes and the corresponding

sample matrix, and sensitivity. Consequently, we discuss several outstanding properties

of the nano-immunosensors and their research opportunities as well as the development

potential and prospects. Also, we summarize below examples of nanostructure material

applications in electrochemical immunosensing biomarkers in whole blood reported so

far in the literature, with their advantages and limitations, and we stress their potential for

future development in this field.

To the best of our knowledge,, current methods for the quantitative detection of

antibodies, including ELISAs, Western blots, and fluorescence polarization assays, are

complex, multiplestep processes that rely on well-trained technicians working in well-

equipped laboratories [24]. In response, researchers would like to find some of other cost

effective an -

coworkers [25] described a versatile, DNA-b c c c “ w c ”

single-step measurement of specific antibodies directly in undiluted whole blood. The

design of this switch takes advantage of the occurrence of two antigen-binding sites on

each antibody, which are separated by ∼12 nm [26]. Specifically, these researchers used

DNA to engineer a switch that brings into close proximity (<4 nm) two copies of an

g ( c “ g ” or simplicity), via the formation of

a stem-loop structure. The two antigens, both linked at the extremities of the DNA strand

are thus located in the middle of the two strands of the stem (Figures 1 and 2). Upon

binding of the antibody to one of these antigens, the high effective concentration of the

second antigen provides the driving force to open the switch (more favorable bidentate

binding. [27] Thus separate the reporter elements. These researchers motivation for using

DNA as the scaffold for this switch is threefold. First, the chemistry of DNA supports the

addition of a variety of antigens ranging from small molecules to polypeptides and

proteins, either during its automated chemical synthesis or through postsynthesis

conjugation. [28] Second, DNA based switches are robust (i.e., they are not triggered by

nonspecific interactions) and relatively stable against degradation. Finally, the base-

g c DNA y w c ’ y c

the sensor achieves optimal detection limits [29].

Also, Romania researchers proposed a novel tool for screening of whole blood for early

detection of breast cancer antigen (CA153) [30]. In this report, Stefan-van Staden and

coworkers propose a stochastic microsensor based on maltodextrin with dextrose

equivalent between 4 and 7. Maltodextrin has a compact helix structure. Materials such

as maltodextrins were excellent candidates for the design of stochastic microsensors used

in the screening of whole blood for breast cancer biomarker. The results obtained for the

assay of breast cancer antigen in whole blood using this microsensor (by applying a

potential of 0.125 V vs. Ag/AgCl and performing measurements specific to stochastic

sensing described in detail in the article) were in agreement with those obtained using the

standard method, ELISA (Enzyme-Linked Immunosorbent Assay utilized a monoclonal

anti-CA15-3 antibody directed against intact breast cancer antigen, CA 153 for solid

phase immobilization on the microtiter wells.. The limit of determination obtained for

breast cancer antigen (0.5mU/mL) when assayed in whole blood, proved that the

microsensor is a good tool (in terms of sensitivity, selectivity, and fast response) for early

screening of whole blood for breast cancer.

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At the similar report by these Romania researchers [31], proposed six new stochastic

microsensors based on physical immobilization of oleoylethanolamide (1), (Z)- N-[(1S)-

2-hydroxy-1-(phenylmethyl)ethyl]-9octadecenamide (2), Nphenethyloleamide(3), N-[2-

(4-methoxyphenyl)ethyl]oleamide(4), N-[1]naphthyloleamide (5), N-cyclohexyloleamide

(6) in graphite paste, for the assay of carcinoembryonic antigen in the human whole

blood. According to obtained result by these researchers, the most sensitive microsensors

were based on oleamide 4, and oleamide 1, respectively immobilized in graphite paste.

The microsensors based on oleamide 1 and oleamide 4 were also presenting the lowest

limit of determination (0.1 pg/mL).

Literature review show that these researchers have another reports in design of stochastic

sensors for electrochemical biomarkers in whole blood.[32-34]

Recently, having the advantages of easy operation, fast response and low cost,

electrochemical technique based detection systems have also gained much attention for

cancer cell detection with high sensitivity even at the single cell level [35, 36].

For this purpose, Liu et al [37] developed an electrochemical impedimetric biosensor for

rapid, sensitive, and low-cost detection of cancer cells at low concentration in blood by

taking advantage of the high selectivity and affinity of antibodies as well as the large

surface and good conductivity of multi-walled carbon nanotubes (MWCNTs). As shown

in Fig. 3 the multilayers of MWCNTs with the negatively charged carboxylate groups are

assembled on indium tin oxide (ITO) glass with the positively charged polyelectrolytes

by the layer-by-layer (LBL) assembly technique. The antibodies of epithelial cell-specific

markers, EpCAM antibodies, are then linked to MWCNTs in the multilayers via a

carbodiimide-mediated wet-chemistry approach. The resulting EpCAM

antibodies/MWCNT nanocomposites are used as nanoscale anchorage substrates to

effectively capture cells on the electrode surface via the specific binding between cell

surface EpCAM and EpCAM antibodies. The EpCAM expressed human liver cancer cell

(HepG2) is used as a model for circulating tumor cells, while the human cervical

carcinoma cell line (HeLa cells) and normal human hematologic cells, which do not

express EpCAM, are used as mixed cells to interfere with detection of HepG2 cells. The

electrochemical impedance of the prepared biosensors is linear with the logarithm of

concentration of the liver cancer cell line (HepG2) within the concentration range of 10 to

105 cells per mL. The detection limit for HepG2 cells is 5 cells per mL. The proposed

method by these researchers is confirmed to be simple, rapid and sensitive for real-time

measurement of the target cells in blood specimens at low concentration.

Label-free nanosensors can detect disease markers to provide point-of-care diagnosis that

is low-cost, rapid, specific and sensitive [38, 39]. However, detecting these biomarkers in

physiological fluid samples is difficult because of problems such as biofouling and non-

specific binding, and the resulting need to use purified buffers greatly reduces the clinical

relevance of these sensors. Hence, to overcome this limitation, researchers would like to

explore novel methods such as using distinct components within the sensor to perform

purification and detection.

For example, a novel label-free electrochemical aptasensors for thrombin detection in

whole blood using self-assembled multilayers with carboxymethyl-PEG-carboxymethyl

(CM-PEG-CM) and thrombin-binding aptamer (TBA) was developed [40]. In the sensing

strategy, CM-PEG-CM and TBA were assembled on the electrode surface via covalent

binding. In the presence of target, the TBA on the outermost layer of the self-assembled

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multilayer would catch the target on the electrode interface, which makes a barrier for

electrons and inhibits the electro-transfer, resulting in the decreased DPV signals. Using

this strategy, a wide detection range (1 pM-160 nM) for target thrombin was obtained,

with a low detection limit of 1.56×10-14

M. The control experiments were also carried out

by using bull serum albumin (BSA) and lysozyme in the absence of thrombin. The results

showed that the aptasensors had good specificity, stability and reproducibility to

thrombin. Moreover, the aptasensors could be used for detection of thrombin in whole

blood which could provide a promising platform for fabrication of aptamer based

biosensors in clinical application.

Also, an electrochemical Lab-on-a-Disc (eLoaD) platform for the automated

quantification of ovarian cancer cells (SKOV3) from whole blood was reported by

Nwankire and coworkers [41]. This centrifugal microfluidic system combines complex

sample handling, i.e., blood separation and cancer cell extraction from plasma, with

specific capture and sensitive detection using label-free electrochemical impedance. Flow

control is facilitated using rotationally actuated valving strategies including siphoning,

capillary and centrifugo-pneumatic dissolvable film (DF) valves. For the detection

systems, the thiol-containing amino acid, L-Cysteine, was self-assembled onto smooth

gold electrodes and functionalized with anti-EpCAM. By adjusting the concentration of

buffer electrolyte, the thickness of the electrical double layer was extended so the

interfacial electric field interacts with the bound cells. Schematic and image sequence of

the full assay protocol was shown in Fig. 4. Significant impedance changes were

recorded at 117.2 Hz and 46.5 Hz upon cell capture. Applying AC amplitude of 50 mV at

117.2 Hz and open circuit potential, aminimum of 214 captured cells/mm2 and 87%

capture efficiency could be recorded. The eLoaD platform can perform five different

assays in parallel with linear dynamic range between 16,400 and (2.670.0003)106 cancer

cells/mL of blood, i.e. covering nearly three orders of magnitude.

Biomarker detection based on nanowire biosensors has attracted a significant amount of

research effort in recent years. However, only very limited research work has been

directed toward biomarker detection directly from physiological fluids mainly because of

challenges caused by the complexity of media. This limitation significantly reduces the

practical impact generated by the aforementioned nanobiosensors. Recently, Chang and

coworkers [42] demonstrate an In2O3 nanowire-based biosensing system that is capable

of performing rapid, label-free, electrical detection of cancer biomarkers directly from

human whole blood collected by a finger prick (Fig. 5). Passivating the nanowire surface

successfully blocked the signal induced by nonspecific binding when performing active

measurement in whole blood. Passivated devices showed markedly smaller signals

induced by nonspecific binding of proteins and other biomaterials in serum and higher

sensitivity to target biomarkers than bare devices. The detection limit of passivated

sensors for biomarkers in whole blood was similar to the detection limit for the same

analyte in purified buffer solutions at the same ionic strength, suggesting minimal

decrease in device performance in the complex media. We then demonstrated detection

of multiple cancer biomarkers with high reliability at clinically meaningful

concentrations from whole blood collected by a finger prick using this sensing system.

To complete our discussion in this sub-section, Table 2 summarizes recent reports on

analytical parameters of some electrochemical immunesensing methods in unprocessed

whole blood.In this sub-section some of nanomaterial-based electrochemical

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immunosensors in whole blood are comprehensively summarized. More importantly,

different aspects of the immunosensors such as type of nanomaterials, injection and

detection techniques, labels, analytes and the corresponding sample matrix, the

sensitivity, etc. are discussed in detail. Consequently, several outstanding properties of

the whole blood immunosensors and their research opportunities as well as the

development potential and prospects are discussed. Additionally, we summarized

examples of nanomaterial based electrochemical immunosensors reported so far in the

literature, with their advantages and limitations and stress their potential for future

developments in this field.

2.3. Other paper of interest

Other than the above discussed nanomaterial-based electrochemical biosensors, in the

literature, other types of nanomaterial-based design electrochemical biosensors for

different types of analytes have been reported. In the current sub-section, we will briefly

discuss some of these nanobiosensors. In this sub-section of review, we focused our

attention on neurotransmitters, proteins and biomarkers biosensing in whole blood.

Despite their easy realization, for amperometric/voltammetric biosensors, few research

efforts have been devoted to these electrochemical nanobiosensors. Nevertheless, there is

a few numbers of promising works concerning o neurotransmitters, proteins now

available in the literature.

Neurotransmitters are the brain chemicals that are released from nerve cells which allow

At the present sub-section, more recent researches on electrochemical biosensing of

neurotransmitters in whole blood based on nanomaterials were review.

Dopamine (DA), epinephrine (EP) and norepinephrine (NE) are very important

neurotransmitters in the mammalian central nervous system, and they play an essential

role in the regulation of physiological processes in living systems. In the case of detection

of DA, EP and NE in whole blood, Stefan-van Stadena and coworkers [43] developed a

new multimode sensors for pattern recognition of neurotransmitters: DA, EP and NE in

biological fluids such as whole blood and urine with aminimum sampling of the

biological fluid (e.g., mixture of the biological fluid with an optimized buffer of pH 3.01

in a ratio1:1 (v/v)). In this report, diamond paste matrix was modified with three different

porphyrins: 5,10,15,20-tetraphenyl-21H, 23H-porphyrine,hemin and protoporphyrin IX

in order to design the multimode sensing systems. Stochastic mode represents a new

approach of electroanalysis and is the best for qualitative analysis using electrochemical

sensors; it can be used reliable also for quantitative analysis. In this report, the lowest

limits of quantification were: 10-10

mol/L for dopamine and epinephrine, and 10-11

mol/L

for norepinephrine. Also, obtained results by this group show that, the multimode

microsensors were selective over ascorbic and uric acids and the method facilitated

reliable assay of neurotransmitters in urine samples, and therefore, the pattern recognition

showed high reliability (RSD<1% for more than 6 months) for the simultaneous

determination of dopamine, epinephrine and norepinephrine from urine and whole blood

samples.Arizona State University researchers published a new article concerning

biosensing of neurotransmitters in whole blood based on nanomaterials. Dai et al [44]

utilized low-cost mesoporous carbon inks to screen print single-use disposable electrodes

for the detection of norepinephrine amperometrically. There are numerous advantages to

amperometric sensing in terms of cost, simplicity and maturity of the technologies.

However, the low concentration (90-220 pg/mL) of NE in human blood provides a

Page 12 of 27

13

significant challenge to obtaining acceptable signal to noise when only the current is

being measured to assess the concentration in blood. To overcome the lower

concentration limits for the amperometric sensing, these researchers increased the surface

area of the electrode to enhance the current signal by use of triconstituent assembly and

silica etching; surface areas from 1500 to 2300 m2/g can be produced depending upon the

carbon-silica ratio during self-assembly. To assess the role of the small micropores, one

control material without silica is also examined. The performance of these screen printed

mesoporous carbon electrodes is strongly dependent upon the surface area with greatest

sensitivity for the highest surface area carbon. To ensure NE can be effectively oxidized,

an enzyme, phenylethanolamine N-methyl transferase, with a cofactor, S-(5ˊ-Adenosyl)-

l-methionine chloride dihydrochloride, to active the enzyme is used to catalyze the

reaction. The resultant sensors can detect NE at concentrations as low as 100 pg/mL-1

in

rabbit whole blood.While highly sensitive protein detection methods have been achieved

in serum samples, biomarker detection in unprocessed whole blood remains very

challenging due to non-specific binding of cells and particulates to the sensor surface.

One approach to perform biosensing in whole blood samples has been demonstrated by

Stern et al., [45] who have developed a microfabricated microfluidic purification chip to

c 10μL w b 20 H w v c q e only achieves

500pM sensitivity, which is 106 times less sensitive than the techniques established for

detection in serum. Moreover, this level of sensitivity is not pertinent to most protein

biomarkers, whose serum concentrations in healthy individuals range between 0.01-100

pM. Hence, researchers would like to explore some of high efficient method for detection

of proteins.

For example Taipei Medical University researchers evaluated an electrochemical

biosensor for uric acid measurement in human whole blood samples [46]. In this work, a

commercially available uric acid monitoring system that is chemically modified to reduce

interference was evaluated via clinical evaluation for its performance and interference as

compared to a centralized laboratory instrument.

There has been significant progress over a period of two decades to develop

electrochemical-sensing approaches for heparin. The early breakthroughs were by

Meyerhoff and Yang, which used liquid PVC-based membranes doped with adequate

sensing components for the direct detection of heparin and protamine in blood [47]. The

strong protamine-heparin interaction makes it possible to use a protamine-selective

electrode in heparin assays, which also minimizes possible biases arising from weaker

nonspecific interactions of heparin with other blood components. Unfortunately,

spontaneous extraction of the polyion into the membrane made these types of sensors

operationally irreversible.

In subsequent years, a number of groups explored dynamic electrochemistry-based

methods to render the heparin or protamine detection operationally reversible. [48]

Shvarev group used mainly controlled current techniques for this purpose. While

protocols mainly useful as titration end points were successful early on, [49] linear

calibration curves less dependent on the background electrolyte of the sample were

achieved with chronopotentiometry. [50] An improved membrane formulation with a

selective membrane containing an excess of the protamine recognizing anion

dinonylnaphthalene sulfonate (DNNS) was recently shown to exhibit the required

Page 13 of 27

14

sensitivity and selectivity as well as linear calibration curves for use in whole blood

samples. [51]

Thin layer coulometry with ion-selective membranes has recently been explored in

Bakker group for the detection of a variety of ions [52, 53]. While a thin layer allows one

to work with just a few microliters of sample, coulometry is a very promising

interrogation platform since the absolute counting approach reduces the need for frequent

calibration. The present work aims to dramatically reduce the required sample volume

and to introduce a heparin sensing principle that is potentially calibration free.

Crespo and corkers [54] explore a potentially calibration-free methodology for the

detection of protamine (and, by titration, heparin) in whole of human blood in the

therapeutic concentration range from 20 to 120 mg/L. The use of a thin layer sample (5.8

μL) c b w b c v b Ag/AgC w

achieves an exhaustive depletion from the sample. In this report, coulometry detection

was chosen for the interrogation of the thin layer, employing a double pulse technique

with 120 s for each pulse. Protamine calibration curves were recorded at physiological

concentrations and in undiluted human blood. Heparin-protamine titrations were

performed in undiluted human blood samples, mimicking the final application with

patients undergoing critical care. The observed values correlate satisfactorily with those

of an alternative technique, so-called flash-chronopotentiometry on planar membranes.

Researches of this work, believed that further progress should involve (1) an

improvement of the biocompatibility of the inner electrode element in contact with blood,

(2) the microfabrication of a precise and accuracy thin layer compartment accompanied

with a tuning of the electrochemical protocol to reduce the intercept of the calibration

curve as much as possible, and (3) replacing the inner liquid solution by a solid ion-to-

electron transducer. Further, such progress may result in a point-of-care analysis system

for heparin that will be as simple to use as an off-the shelf glucose test strip.

3. Conclusions and future prospects

Nanomaterial-based electrochemical (bio/immune)-sensors will play an increasing role in

electroanalytical science in the near future. For electroanalytical applications, research is

required into the development of protocols for synthesis and functionalization of

nanomaterials. For bioanalytical applications, research should focus on increasing the

sensitivity and the selectivity of the nanobiosensors and on lifetime-based detection

methodologies. The fast advancement of nanomaterial-based electrochemical

(bio/immune)-sensors highlights their future applications in diverse scientific fields. The

major impacts appear to be in early detection of diseases, genetic mutations and

biotargets. Electrochemical (bio/immune)-sensors based nanomaterial provide fast,

simple, sensitive detection systems for cancer that may provide robust tools for

anticancer biosensor research. Despite significant advances in electrochemical

(bio/immune) based on nanomaterials, there are still challenges to explore new protocols

and strategies for improving the sensitivity and practical applications of the

electrochemical (bio/immune), as follows:

(1) Until now, the notable characteristics of electrochemical (bio/immune) sensing (e.g.,

disposable electrode array, label-free, multiplex analysis and microfluidic flow injection)

were rarely mentioned in whole/unpreceded blood. These characteristics indicate the

great opportunities for researchers in these scientific disciplines. The intrinsic

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electrochemical properties of nanomaterials would provide excellent theoretical support

for the development of label-free electrochemical immunosensors.

(2) Results raise doubts about repeatability, reproducibility, and comparability of

nanomaterials based-electrochemical (bio/immune)-sensing; as with every emerging

technology, standards need to be established to avoid doubts about the lack of

reproducibility, repeatability, and compatibility across platforms and laboratories.

(2) There is no comparison of the robustness of the nanomaterials for electrochemical

cytosensing in the literature. Obviously, electrochemists have a great deal to do to

address the performance of nanomaterials based-electrochemical (bio/immune)-sensing

in the future.

(3) Designing methodology for proper immobilization of cells on a nanomaterials based

compounds is a complex mixture of sciences and arts. There are often conflicting

requirements from stability and activity. Diversity of the process conditions necessarily

requires the design of specific, immobilized cells that can match the corresponding

requirements for the desired application.

(4) Another puzzling challenge is to develop a novel, simple method for immobilizing

enzymes on nanomaterials to reduce further electron-transfer resistance and to improve

stability, sensitivity, selectivity and life-span of electrodes.

(5) The long-term goals associated with incorporating nanomaterials into cytosensing

technology suggest that further research is required before these devices reach sufficient

performance standards.

(6) The use of nanomaterial tags for detecting proteins, neurotransmitters is still in its

infancy. Due to the minority of research being on development new nanomaterial-based

electrochemical biosensors for detecting proteins, neurotransmitters n whole blood , more

electrochemical techniques should be involved in this area. With nanomaterial, it will be

possible for biosensors to be applied to pre-warning and real-time detection of diseases.

(7) Unfortunately, minor reports (discussed above) can be seen to apply label-free

methods for electrochemical nanobiosensing. But, above examples shows that label-free

methods play a key role in manipulating various intracellular processes for

electrochemical biosensing applications. In general, until now, the remarkable

characteristics of label-free electrochemical biosensors were rarely mentioned. These

remarkable characteristics nicely indicate the great opportunities for researchers. The

intrinsic electrochemical properties of label-free electrochemical biosensors will provide

excellent theoretical support for the development of these biosensors.

(8) The biofouling of electrode surface will bring catastrophic damage to the electron

transfer between enzyme and electrode redox center. Therefore researchers should be

solving this problem using application of novel nanomaterials on structure of

electrochemical biosensors.

From the above examples, we conclude that there is a bright opportunity for further

advances and developments of nanomaterials-based (bio/immune)-sensing devices based

on electrochemical methods, especially through further miniaturization and integration

into lab-on-chip systems. The design of implantable (bio/immune)-sensing with the

ability to in vivo detection of cells and real time analysis is promising for the application

of electrochemical (bio/immune)-sensors, even though it is yet to be explored. Therefore,

electrochemists have a great deal to for to address electrochemical (bio/immune)-sensors

behavior based on nanomaterials in the future. In general, electrochemical (bio/immune)-

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sensors based on nanomaterials show great promise for future applications in health-care

testing and disease diagnostics. Given the impressive progress in nanomaterials-based

electrochemical systems, there is no doubt that they will have major impact on POC

clinical diagnostics. In future research, special attention will probably be given to

implantable electrochemical (bio/immune)-sensors based on nanomaterials for detection

and determination several of substance such as protein, glucose, inorganic salt, hormone,

biomarkers and so on. Future research will most probably explore the use of combination

nanomaterials with biomarkers, proteins, enzymes and so on in order to improve the

performance of implantable electrochemical immunosensors.

ACKNOWLEDGMENTS

Partial support through Drug Applied Research Center, Tabriz University of Medical

Sciences and Nano Technology Center, Faculty of Chemistry, Urmia University are

greatly acknowledged.

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Figure legends:

Fig. 1. Antibody (Ab)- c v c c c “ w c ” [41

Fig. 2. Antibody-activated switch architecture, [41].

Fig. 3: Preparation of ITO/MWCNT/PEI/anti-EpCAM and immunological recognition

between anti-EpCAM and HepG2 [68].

Fig. 4: Schematic (a-e) and image sequence (i-v) of the full assay protocol for integrated

centrifugal microfluidic platform [73].

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nanosensor in buffer, (b) unpassivated CA-125 nanosensor in serum [74].

Table 1: Analytical parameters of some electrochemical enzyme nanobiosensing

methods in unprocessed whole blood

Type of electrode modifier Type of

analyte

Detection potential

versus Ag/AgCl

LOD Ref

carboxymethyl-PEG-

carboxymethyl (CM-PEG-CM)-

GCE

glucose -0.29 V 12.4 µM 10

polyurethane-Pluronic F127

(PUF127) nanospheres and

immobilizing glucose oxidase

(GOx) on (PU-F127)-glass

carbon electrode (GCE)

glucose -0.49 V 11.4 µM 11

graphene/polyaniline/Au

nanoparticles/glucose

oxidase(Gr/PANI/AuNPs/GOD)-

SPCE

glucose - - 13

µ-PADs (S-layer protein SbpA

(from Lysinibacillus sphaericus

CCM 2177))

glucose - 6.4 mM 13

ePADs (GOx-Prussian blue

modified screen printed

electrode (PB-SPEs).

glucose -0.1 V 3.4 mM 18

Au nanoparticles-H3O-SO3H

nanoparticles -glucose oxidase

(GOx) modified glassy carbon

electrode (GCE)

glucose - 12 µM 22

Page 20 of 27

21

Table 2: Analytical parameters of some electrochemical immunesensing methods in

unprocessed whole blood

Type of electrode modifier Type of analyte Detection

potential

versus

Ag/AgCl

LOD Ref

stochastic microsensor based on

maltodextrin with dextrose

equivalent between 4 and 7

breast cancer

antigen- CA 153

0.125 V 0.5mU/mL 30

stochastic microsensors based on

physical immobilization of

oleoylethanolamide (1), (Z)- N-

[(1S)-2-hydroxy-1-

(phenylmethyl)ethyl]-

9octadecenamide (2),

Nphenethyloleamide(3), N-[2-(4-

methoxyphenyl)ethyl]oleamide(4),

N-[1]naphthyloleamide (5), N-

cyclohexyloleamide (6) in

graphite paste

carcinoembryonic

antigen

- 0.1 pg/mL 31

stochastic microsensors based

Mn(III) with meso-tetra (4-

carboxyphenyl) porphyrin, and

maltodextrin (dextrose

equivalence between 4 and 7),

immobilized in diamond paste,

graphite paste or C60 fullerene

paste

HER-1 - 280 fg/ml

and 4.86

ng/ml

32

stochastic microsensors based on

M α-cyclodextrin and

5,10,15,20-tetraphenyl-21H,23H

leptin 1.25 × 10-10

33

plasminogen

activator

1 × 10-12

Page 21 of 27

22

porphyrin were immobilized in

carbon based matrices such as

diamond paste, graphite,

graphene, carbon nanotubes, and

C60 fullerenes.

inhibitor-1 (PAI-

1)

stochastic microsensors based four

electrochemical active materials:

α-cyclodextrin, maltodextrin with

dextrose equivalent, the complex

of Mn(III) with 5,10,15,20-

tetraphenyl-21H,23H-porphyrin,

and the traditional hemolysine

immobilized in diamond paste

matrices

hepatitis B - - 34

Cytosensor: EpCAM

antibodies/MWCNT

nanocomposites

HepG2 cells - 5 cells per

mL

37

self-assembled multilayers with

carboxymethyl-PEG-

carboxymethyl (CM-PEG-CM)

and thrombin-binding aptamer

(TBA)

thrombin - 1.56×10-14

M

40

Lab-on-a-Disc (eLoaD) platform

based on thiol-containing amino

acid, L-Cysteine, was self-

assembled onto smooth gold

electrodes and functionalized with

anti-EpCAM

ovarian cancer

cells (SKOV3)

- 214

captured

cells/mm2

41

Page 22 of 27

23

Fig. 1

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24

Fig. 2

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Fig. 3

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Fig. 4

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Fig. 5

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