Biosensors and Bioelectronics 76 (2016) 80–90

Contents lists available at ScienceDirect

Biosensors and Bioelectronics

journal homepage: www.elsevier.com/locate/bios

Engineering the bioelectrochemical interface using functional

nanomaterials and microchip technique toward sensitive and portable
electrochemical biosensors
Xiaofang Jia, Shaojun Dong, Erkang Wang n
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China

art ic l e i nf o a b s t r a c t

Article history: Electrochemical biosensors have played active roles at the forefront of bioanalysis because they have the
Received 30 March 2015 potential to achieve sensitive, specific and low-cost detection of biomolecules and many others. En-
Received in revised form gineering the electrochemical sensing interface with functional nanomaterials leads to novel electro-
13 May 2015
chemical biosensors with improved performances in terms of sensitivity, selectivity, stability and sim-
Accepted 14 May 2015
Available online 15 May 2015
plicity. Functional nanomaterials possess good conductivity, catalytic activity, biocompatibility and high
surface area. Coupled with bio-recognition elements, these features can amplify signal transduction and
Keywords: biorecognition events, resulting in highly sensitive biosensing. Additionally, microfluidic electrochemical
Electrochemical biosensors have attracted considerable attention on account of their miniature, portable and low-cost
Biosensors
systems as well as high fabrication throughput and ease of scaleup. For example, electrochemical en-
Nanomaterials
zymetic biosensors and aptamer biosensors (aptasensors) based on the integrated microchip can be used
Microfluidic chip
Aptamer for portable point-of-care diagnostics and environmental monitoring. This review is a summary of our
Cytosensor recent progress in the field of electrochemical biosensors, including aptasensors, cytosensors, enzymatic
Enzyme biosensors and self-powered biosensors based on biofuel cells. We presented the advantages that
Biofuel cell functional nanomaterials and microfluidic chip technology bring to the electrochemical biosensors, to-
gether with future prospects and possible challenges.
& 2015 Elsevier B.V. All rights reserved.

1. Introduction optical properties of metal nanoparticles (NPs) (Mayer and Hafner,

2011), electrical conductivity of carbon nanomaterials (Guo and
Electrochemical biosensor is a device transducing biological Dong, 2011), electrocatalytic properties of metal NPs and nano-
sensing element-target recognition events into detectable elec- carbons (Banks and Compton, 2005), and high surface area. Cou-
trochemical signals (Turner et al., 1987). Biological sensing ele- pling functional nanomaterials, especially the carbon and metal
ments (e.g., enzymes, aptamers or antibodies) are immobilized/ nanomaterials, with bioelectrochemical sensing interface, injects
integrated at the electrochemical interface. Upon target binding, fresh energy to the development of electrochemical biosensors
the electrochemical signal of redox reporter is changed, which is (Guo and Dong, 2009; Guo and Wang, 2011). The advantages that
directly related to the concentration of target species. Electro- nanomaterials bring to electrochemical biosensors are presented
chemical biosensors have been attracting much attention owing to as follows but not limited to: enlarging the electrochemically ac-
their simple configurations, low cost, multiplexed detection cap- tive areas, accelerating electron transfer between electrodes and
abilities, high sensitivity and selectivity, as well as ease of minia- detection species, and behaving as biocompatible scaffolds for
turization for portable point-of-care diagnostics and environ- biomolecule immobilization. These remarkable features lead to
mental monitoring (Kimmel et al., 2012; Privett et al., 2010). novel electrochemical biosensors with better performances such
Engineering the bioelectrochemical sensing interface is crucial as improving the sensitivity and stability.
for improving the sensitivity and stablility of electrochemical With the rapid development of chemical/biosensor networks
biosensors. Nanomaterials display many unique properties that (Byrne and Diamond, 2006) and growing concern about health-
are dependent on their sizes and shapes, such as, size-dependent care, food safety and environment pollution, fast access to bio
(chemical) information is highly in demand. Therefore, it is of
n
Corresponding author. paramount importance to develop low-cost point-of-care diag-
E-mail addresses: [email protected] (S. Dong), [email protected] (E. Wang). nostics and environmental monitoring devices. Fueled by the

http://dx.doi.org/10.1016/j.bios.2015.05.037
0956-5663/& 2015 Elsevier B.V. All rights reserved.
 X. Jia et al. / Biosensors and Bioelectronics 76 (2016) 80–90 81

urgent need, researchers turn their attention to microfluidic

electrochemical biosensors due to their miniature, portable and
low-cost systems as well as high throughput and automation.
In this review, we summarized our recent advances in en-
gineering the bio-nano sensing interface for sensitive and portable
electrochemical biosensors. Our work covered four kinds of elec-
trochemical biosensors, involving aptamer biosensors (apta-
sensors), cytosensors, enzymatic biosensors and self-powered
biosensors based on biofuel cells (BFCs). We presented the ad-
vantages that nanomaterials and microfluidic chip technology
bring to the electrochemical biosensors.

2. Electrochemical aptasensors

Since firstly discovered by three groups independently in 1990

(Ellington and Szostak, 1990; Robertson and Joyce, 1990; Tuerk and
Gold, 1990), aptamers have aroused enormous interest in the sci-
entific community. Aptamers are artificial single-stranded DNA,
RNA or peptide sequences. Upon folding into unique secondary
and tertiary structures, aptamers are capable of adaptively binding
to certain targets with high specificity and affinity (Mascini et al.,
2012). They are isolated from a library pool with
1016 random
nucleic acid sequences through in vitro selection process known as
systematic evolution of ligands by exponential enrichment (SE-
LEX). The targets of aptamers are quitely wide from small mole-
cules to proteins and even whole cells, with binding affinities Fig. 1. The schematic representation of EIS aptasensors. (A) Circle model for the EIS
and the schematic of EIS aptasensor for adenosine based on the duplex-to-complex
comparable to or even stronger than that of antibodies (Kds from
design. (B) The sensing strategy for EIS detection of ATP. Without target, graphene
picomolar to nanomolar level) (Famulok and Mayer, 2011; Iliuk was absorbed on ABA modified Au electrode, resulting in a decreased Rct. In the
et al., 2011). Besides the merits mentioned above, aptamers are presence of target, the formed duplex could not adsorb graphene, leading to the
produced in vitro, making it easier to tailor them with functional restoration of the high Rct. The developed strategy was also applicable for the de-
groups for diverse applications. Also, aptamers are more stable tection of Hg2 þ . Adapted from B. Li et al. (2007) and Wang et al. (2012) with
permission.
than antibodies and can endure harsh conditions such as high
temperature or extreme pH. These features make aptamers more
competitive as recognition elements compared with antibodies for decreased Rct. The EIS method provided a sensitive detection for
a variety of applications in the fields of biosensing, biomedicine adenosine with the detection limit of
10  7 M. Additionally, the
and cell biology. designed aptasensor can be easily regenerated by hybridizing ABA
Up to date, a great deal of electrochemical aptasensors have with free PCS on the electrode. Subsequently, the α-thrombin
been developed, most of which required the step of labeling ap- binding aptamer (TBA) was grafted onto the ABA strand, achieving
tamers or other nucleic acids with the redox reporters (Li et al., the parallel detection of ATP and α-thrombin (Du et al., 2008).
2010; Liu et al., 2011; Lubin and Plaxco, 2010). The labeling process Addition of α-thrombin amplified the sensitivity of ATP molecule,
suffers from cost- and labor-intensive, and to some extent redu- and the sensing surface was directly regenerated by treating with
cing the binding affinity of the aptamers to their targets. In re- a large amount of ATP.
sponse, our group is devoted to the exploration of “label-free” For the EIS aptasensors for protein, the signal heavily relied on
strategies without the modification of “special electrochemical the size or charge of protein, limiting the detection sensitivity (Xu
probes”. et al., 2005). To solve this problem, we fabricated a sandwich
sensing platform in which the thrombin molecules were captured
2.1. Electrochemical impedance technique by the thiolated aptamers immobilized on the Au electrode, and
then the aptamer functionalized AuNPs were used to amplify the
Electrochemical impedance spectroscopy (EIS) has been re- impedimetric signals (Li et al., 2008). Through the amplified sen-
cognized as a sensitive tool for probing the interfacial properties of sing strategy, a very low detection limit of 0.02 nM was realized for
surface-modified electrodes (A.X. Li et al., 2007). When the targets the detection of thrombin.
bind with aptamers at the electrode surfaces, the capacitance and Graphene is a two-dimensional carbon material with single-
interfacial electron transfer resistance of the electrodes are chan- atom-thick. It possesses the advantages of large surface area and
ged. This provides a convenient method to construct highly sen- excellent electron conductivity (Fang and Wang, 2013; Guo and
sitive and cost-effective label-free aptasensors. Dong, 2011), which provides new opportunity for developing more
Early in 2007, we developed an EIS aptasensor for the detection sensitive EIS aptasensors. As shown in Fig. 1B, through strong π–π
of adenosine on the basis of duplex-to-complex design (B. Li et al., stacking interaction between nucleotide and graphene, graphene
2007). As depicted in Fig. 1A, the adenosine-binding aptamer was absorbed on the ssDNA ABA modified Au electrode, resulting
(ABA) strand was partially hybridized with 5'-thiolated part in largely decreased Rct (Wang et al., 2012). Upon addition of ATP,
complementary strand (PCS). The formed self-assembly layers ABA underwent a conformational alteration to form duplex
with negative charge repulsed the negatively charged probe strands. It led to the desorption of graphene and the restoration of
[Fe(CN)6]4  /3  anions, and hindered interfacial electron-transfer high Rct. On the basis of this, a sensitive EIS aptasensor was fab-
kinetics of the redox probes with high charge transfer resistance ricated for the detection of ATP with the detection limit down to
(Rct). Upon addition of adenosine, the amount of the aptamers on 15 nM. The proposed detection strategy was versatile, which was
the electrode surface was reduced, accompanied with the demonstrated by the detection of Hg2 þ using the Hg2 þ -specific
82 X. Jia et al. / Biosensors and Bioelectronics 76 (2016) 80–90

oligonucleotide probe. Based on π–π stacking interaction of nu- targets such as proteins, DNA, or drugs were present, they inter-
cleotide and graphene, we also fabricated a label-free EIS aptamer acted with aptamers and led to sluggish or accelerated electron
sensing interface for the detection of α-thrombin. This design to- transfer kinetics, which was sensitively probed with the DPV sig-
tally removed the need of labeling the aptamers with functional nal of the probe Fc. The “solid-state” electrochemical technique
groups (  SH,  NH2), which was generally used in the fabrication can greatly improve the analytical performance of aptasensors
of the sensing interface process (Wang et al., 2011). The sensing such as sensitivity, selectivity and applicability.
interface was fabricated by adsorbing TBA on the graphene layer For example, using the graphene–mesoporous silica–gold NP
that was assembled on a positively charged Au film via electro- hybrids (GSGHs) as enhanced element, we fabricated an in-
static interaction. After binding with α-thrombin, TBA was re- tegrated, label-free sensing platform for sensitive and selective
leased from the sensing surface and an obvious Rct decrease was detection of DNA using the strand-displacement DNA poly-
observed. Meanwhile, this designed sensing strategy was also
merization and the parallel-motif DNA triplex system as dual
applicable for surface plasmon resonance aptasensor.
amplification strategy (Fig. 2B) (Du et al., 2011b). Firstly, the added
target DNA strand as a trigger opened the hairpin probe DNA
2.2. “Solid-state” electrochemical technique
template and released the 3' primer binding site. Then the short
primer bound with the template DNA, and triggered the DNA
Our group has been contributing great efforts to developing
polymerization in the presence of DNA polymerase I and deox-
solid-state electrochemiluminescence (ECL) technology by im-
ynucleotide solution mixture (dNTPs), resulting in extensive du-
mobilizing Ru(bpy)32 þ on the electrode surface (Wei and Wang,
2008). Compared with solution-phase ECL, solid-state ECL ex- plex DNA and free target DNA strand. The free target DNA as the
hibited several advantages, such as less or no consumption of catalyst opened the hairpin structure repeatedly and produced
expensive reagent, simplified experimental design and enhance- much dsDNA. Then, the resultant dsDNA was incubated with the
ment of the ECL signal (Li and Wang, 2012). Inspired by this, we sensing interface, and combined with the single-strand acceptor
introduced the concept of “solid-state probe” to the fabrication of DNA on the electrode to form the triplex structure, aided by Ag þ
label-free electrochemical aptasensors using electroactive probe ions. The triplex blocked the electrode surface, leading to a de-
ferrocene (Fc) and differential pulse voltammetry (DPV) technique. creased DPV signal of Fc. Consequently, the DPV signal of Fc de-
As shown in Fig. 2A, the Fc-appended poly(ethyleneimine) (Fc- creased in the target DNA-concentration-dependent manner. A
PEI) complex was firstly synthesized through covalently coupling very low detection limit of 10 fM was obtained, much lower than
the probe Fc with positively charged PEI. Then the Fc-PEI complex other reported graphene-based DNA electrochemical sensors
was immobilized on the ITO array electrode through layer-by-layer (Bonanni and Pumera, 2011). In the sensing process that we de-
(LBL) assembly with negatively charged nanomaterials such as scribed above, the GSGHs possessed several merits for the design
CNTs (Du et al., 2010a), AuNPs (Du et al., 2010b), and graphene (H. of electrochemical DNA sensing platform: (1) the biocompatible
Qin et al., 2012). Aptamers were appended as the outermost layer mesoporous silica reduced the nonspecific adsorption of the DNA;
through the Au–S bond or noncovalent interactions. When the (2) AuNPs facilitated the immobilization of the thioated DNA and
the stability of Fc-PEI assembly through the Au–S and Au–N
bonds; (3) GSGHs had excellent electronic conductivity which was
favorable for the electron transfer of the probe Fc; (4) they greatly
enlarged electrode surface area, which was conducive to signal
amplification.
With additional silver microsphere (SMS)-based separation
step, this sensing system was extended to highly sensitive and
selective detection of ATP (Guo et al., 2011). A prolonged ABA was
covalently immobilized on the SMSs, and hybridized with a
blocker strand. Upon addition of ATP, the blocker strand was re-
leased from the beads. The SMSs with unreacted ABA–blocker
duplex and ATP–ABA complex easily settled down and could be
conveniently removed from the solution, leaving the released
blocker strand in the solution. The amount of blocker DNA was
corresponding to the added ATP. Benefited from the strand-dis-
placement DNA polymerization and the parallel-motif DNA triplex
system dual amplification method, highly sensitive analysis of ATP
was achieved with the detection limit of 0.023 nM.
Based on the GSGHs sensing interface, a facile and sensitive
aptasensor was further developed for distinguishing enantiomers
of arginine vasopressin (VP) using a designed split aptamer con-
taining two fragments P1 and P2 (Du et al., 2012). The fragment P1
as the capture probe was immobilized on the electrode with the
Au–S bond. In the presence of D-VP, the detection probe P2 frag-
ment was in conjunction with the P1 to fold into a three-way
junction, which caused a decreased DPV signal of Fc. The fabri-
cated aptasensor for the detection of D-VP exhibited high sensi-
tivity with a low detection limit of 5 ng mL  1 and high selectivity
Fig. 2. (A) Fabrication method of solid state electrochemical probe based on LBL
towards L-VP. Meanwhile, the designed sensing strategy was ver-
assembly of Fc-PEI and GSGHs. (B) Amplification detection of DNA using the strand-
displacement DNA polymerization and the parallel-motif DNA triplex system dual satile for the detection of other aptamer-based analytes, such as
amplification strategy. Adapted from Du et al. (2011b) with permission. cocaine.
 X. Jia et al. / Biosensors and Bioelectronics 76 (2016) 80–90 83

Fig. 3. Schematic illustration of the sensing procedure for multianalysis of ATP and cocaine using the MECAS Chip: (A) step 1, the sensing interface fabrication; (B) step 2, the
sample inlet and incubation; (C) step 3, the electrochemical detection process. Adapted from Du et al. (2011a) with permission.

2.3. Integrated microchip technique was fabricated for sensitive Hg(II) detection using CC measure-
ments. T-rich Hg2 þ specific oligonucleotide was immobilized on
Recently, electrochemical microfluidic devices have progressed the Au working electrode, which was partially hybridized with a
rapidly because of their ease of miniaturization, high throughput short complementary sequence. Upon incubation with Hg2 þ ions,
and automation. Coupled with the aptamer technique, it is ex- the T-rich sequence folded to a T–Hg2 þ –T mediated hairpin
pected to produce low-cost, convenient, integrated electro- structure and released the short complementary sequence, leading
chemical aptasensors. Our group previously developed a two-step to a decreased CC signal. The detection limit of Hg2 þ was down to
photolithography technique for the fabrication of Au–Ag dual- 1 nM with high selectivity. In combination with home-made USB
metal integrated electrode system (Chen et al., 2010). Utilizing the electrochemical system (μECS), these MECASs have great potential
metal electrode on-chip systems, a microfluidic electrochemical for low-cost portable environmental and healthcare monitoring (Li
aptasensor (MECAS) was constructed for multiplex analysis of et al., 2013).
small molecules (Du et al., 2011a). In our design, all the steps in-
cluding the sensing interface fabrication, sample incubation and
electrochemical detection on-chip were performed in the micro- 3. Electrochemical cytosensors
fluidic channels. As shown in Fig. 3, the MECAS chip was covered
by microfluidic channels PDMS frame 1 (step 1). To fabricate the Despite remarkable advances in our understanding of the dis-
sandwich sensing platform, the ATP aptamer fragment (SH-A2) ease, cancer remains one of the leading causes of death worldwide
and the cocaine aptamer fragment (SH-C2) as capture probes were (Yoo et al., 2011). In 2010, approximately 7.98 million people died
separately modified on two groups of electrodes through the Au–S of cancer, and new cases may further increase to 15 million in the
bond. In step 2, the MECAS chip was covered with the PDMS frame year 2020 according to the World Cancer Report (Y. Liu et al.,
2 vertical to the frame 1, which introduced the sample to two 2013). Developing rapid and sensitive cytosensor for cancer cells is
working electrodes in the same group. The sample contained the in high demand for early detection and treatment. Among various
targets ATP/cocaine and aptamer functionalized AuNPs as the detection techniques, electrochemical methods are easier to min-
amplified element to improve the sensitivity. Finally, the chron- iaturize, and suitable for portable point-of-care testing devices.
ocoulometry (CC) measurements were performed in the PDMS When cancer cells were selectively bound with the recognition
frame 3 (step 3) containing the detection chamber which accom- elements on the electrode, they would result in sluggish electron
modated two groups of three-electrode systems. The electroactive transfer kinetics, which was sensitively monitored with current or
probe [Ru(NH3)6]3 þ was bound to the surface-confined DNA EIS signal change.
through electrostatic interaction. The CC signal linearly increased In 2012, thanks to the signal amplification originated from
with the concentration of target ATP/cocaine. Benefited from mi- electrochemical current rectifier (ECR), we constructed a rapid,
crofluidics technique, we achieved highly sensitive and selective sensitive and non-invasive cytosensor for detection of folate re-
multianalysis of two model small molecules ATP and cocaine ceptor (FR)-rich cancer cells (Li et al., 2012). As a basic molecular
within 40 min. The detection limits of ATP and cocaine were as device, ECR only permits unidirectional current to pass through. As
low as 3  10  10 M and 7  10  8 M, respectively. shown in Fig. 4A, 11-undecanethiol-1-ferrocene and 16-mercap-
Subsequently, we improved the fabrication technique of on- tohexadecanoic acid as redox-active electron transfer mediator
chip Au and Ag electrodes based on different solubility of metal Ag and insulating layer were immobilized on the Au bead electrode,
and Au in KI–I2 and HNO3 solution (Chen et al., 2012). A MECAS which presented a very low current signal. After addition of
84 X. Jia et al. / Biosensors and Bioelectronics 76 (2016) 80–90

Fig. 4. The schematic illustration of electrochemical cytosensor. (A) The cytosensor based on signal amplification from ECR. Cell binding caused a decreased signal. (B) The
ECL cytosensor based on graphene/Fe3O4@luminol-Au multifunctional nanocomposites. Reprinted with permission from Gu et al. (2015) and Li et al. (2012).

K2IrCl6, only unidirectional cathodic current with higher magni- technology to fabricate non-invasive label-free electrochemical cy-
tude was detected. According to the relative redox potentials be- tosensors (J. Liu et al., 2013; Y. Qin et al., 2012). The solid-state
tween surface-confined ferrocene (0.34 V) and iridate (IV/III) ions sensing interface was constructed with LBL assembly of positively
(0.71 V), the electrons were forwarded immediately to the iridate charged Fc-PEI (J. Liu et al., 2013) or Fc functionalized poly(allylamine
(IV) ions, resulting in enhanced cathodic current. Reversely, elec- hydrochloride) (Fc-PAH) (Y. Qin et al., 2012) as the signal indicator
tron transfer from iridate (III) ions to oxidized ferrocene was and negatively charged SWNTs as the signal enhancement element
thermodynamically forbidden, resulting in the disappearance of on Au substrates. Then FA at the outermost layer was covalently
anodic current. HeLa cells were selectively adhered on the elec- bound onto SWNTs as recognition component. The as-prepared
trode through the interaction of target FR on the surface of cancer sensing interfaces exhibited excellent biocompatibility and were
cell and recognition element folic acid (FA). Due to the insulating suitable for cytosensor materials. Using fast-response DPV signal, a
property of cell membrane, electron transfer access was inhibited, wide detection range from 10 to 106 cells mL  1 with a detection
leading to a decreased DPV signal. By virtue of the current mag- limit as low as 10 cells mL  1 was obtained (J. Liu et al., 2013).
nification of the ECR, a broad linear range with the detection limit Recently, we developed a label-free magnetic-control solid-state
as low as 10 cells mL  1 was obtained even in the presence of a ECL sensing platform for detection of HeLa cells using multifunctional
large number of normal cells. In contrast to the commonly used graphene/Fe3O4@luminol-Au nanocomposites (Fig. 4B) (Gu et al.,
EIS technology, fast-response DPV method prevented immobilized 2015). The multifunctional nanomaterials were synthesized through
cells from long exposure to an electric field, allowing the detection assembly of graphene/Fe3O4 hybrids and luminol capped AuNPs
of living cells (Shao et al., 2009). through the Au–N bond and electrostatic adsorption. The aptamer
To develop reliable cytosensor, it is desirable to introduce no AS1411 with specific affinity for nucleolin of HeLa cells surface, was
extraneous reagents that may cause false-positive result. For in- then covalently linked to nanocomposites through the Au–S bond.
stance, enzymatic substrate H2O2, or external redox probes When HeLa cells were selectively captured on the nanocomposites,
Fe(CN)64 /Fe(CN)63 might induce cell apoptosis (Nonn et al., 2003) the interfacial charge transfer kinetics was retarded due to the re-
or unexpected effects on viability of tissue (Pablo et al., 1997), which sistance of the cell membrane, which gave rise to a decreased ECL
have an effect on the detection result. There is a pressing need for signal. The present ECL cytosensor exhibited high sensitivity with a
developing non-invasive cell detection methods. To address this is- detection limit of 8 cells mL  1. The as-prepared multifunctional
sue, we learned from the solid-state electrochemical aptasensors graphene/Fe3O4@luminol-Au nanocomposites owned several unique
 X. Jia et al. / Biosensors and Bioelectronics 76 (2016) 80–90 85

advantages: (1) ECL reagent luminol was directly immobilized on the interesting carbon nanomaterials (Iijima et al., 1999). By laser ab-
nanocomposites without extra additive, endowing the materials with lation of graphite, they were produced with high purity and yield.
good biocompatibility; (2) graphene as the nanocarriers with large In view of impressive properties of SWNHs, Xu's group con-
surface area provided abundant anchoring sites for luminol; (3) AuNPs structed an amperometric glucose biosensor by encapsulating
and graphene with excellent electron conductivity were beneficial for GOD in the Nafion-SWNHs composite film (Liu et al., 2008). The
the electron transfer of luminol; (4) due to the superparamagnetic fabricated biosensor exhibited satisfactory performance with high
property of Fe3O4, the nanocomposites as “dispersible electrodes” sensitivity, stability and selectivity. Then they assembled myoglo-
conveniently captured HeLa cells in solution and then were im- bin with poly(sodium 4-styrenesulfonate) (PSS) modified SWNHs
mobilized on the electrode surface using external magnet-control. for the detection of H2O2, which also displayed good electro-
chemical response (Liu et al., 2010).
Besides the merits mentioned above, we found that graphene
4. Electrochemical enzymatic biosensors with excellent electrocatalytic activity toward H2O2 and NADH,
were used for electrochemical biosensors, exhibiting better ana-
4.1. Amperometric enzymatic biosensors lytical performance for the detection of glucose and ethanol (Zhou
et al., 2009b). Niu et al. found that graphene-PFIL nanocomposites
Enzymatic biosensor was an important branch of electro- exhibited good electrocatalytic activity toward the reduction of O2
chemical biosensors. The determination of biological small mole- and H2O2, resulting from the excellent electron transfer property
cules such as glucose, dopamine and dihydronicotinamide adenine of graphene (Shan et al., 2009). Based on this, they further de-
dinucleotide (NADH), are mainly focused on amperometric enzy- veloped a glucose biosensor by encapsulating GOD in the gra-
matic biosensors. The redox-active center of enzyme is en- phene-PFIL composites. In contrast to the commonly used oxida-
capsulated by the protein layer, which is a barrier of the direct tion current, the reduction current of O2 and H2O2 was used to
electron transfer between redox-active center and the electrode. detect glucose in this method, which eliminated the coexisting
To solve this problem, carbon nanomaterials such as ordered interference of reductive biomolecules such as ascorbic acid and
mesoporous carbons (OMCs), CNTs and graphene, are promising dopamine in the practical clinical analysis. In order to improving
candidates because of their large surface area, excellent electron the analytical performance of graphene-based glucose biosensors,
conductivity and biocompatibility, offering great opportunities to they further constructed graphene/AuNPs/GOD/chitosan compo-
the fabrication of improved electrochemical enzymatic biosensors. sites film modified electrode (Shan et al., 2010b). Both the cathodic
Many studies have demonstrated that CNTs as electrode ma- and anodic currents could be used for glucose detection with
terials can greatly enhance the electrochemical reactivity of NADH, outstanding electrochemical response. They also constructed am-
H2O2 and ascorbic acid (Bai et al., 2012; Wang, 2005), which stems perometric biosensors for the determination of NADH and ethanol
from the presence of high edge plane density on CNTs (Banks and by immobilizing ADH using IL-functionalized graphene and chit-
Compton, 2005). For instance, a sensitive NADH biosensor was osan, which exhibited good analytical performance (Shan et al.,
developed based on CNTs/thionine/AuNPs self-assembled multi- 2010a). Besides, Tian et al. demonstrated that ultrathin graphitic
layers on ITO electrode using thionine as cross-linkers (Deng et al., carbon nitride (g-C3N4) nanosheets, as an analog of graphite, be-
2008). The functional sensing interface not only displayed ex- haved as highly efficient electrocatalyst toward the reduction of
cellent catalytic activity of the oxidation of NADH, but also be- H2O2. They further investigated its application for glucose bio-
haved as the biocompatible scaffold for enzyme immobilization. sensing (Tian et al., 2013).
Interestingly, the photoactive dye thionine allowed reversibly By chemical vapor deposition (CVD) technology, macroporous
tuning the performance of the NADH sensor. In the dark, a de- three-dimensional graphene (3D-G) foam can be prepared with
tection range from 0.5 to 237 μM with the detection limit of large quantity. Relative to 2D graphene, 3D-G possesses superior
0.1 μM and the sensitivity of 17 nA μM  1 was obtained, whereas electrochemical properties, such as rapid charge transfer and mass
the sensitivity improved to 115 nA μM  1 with the detection limit transport kinetics. Using one-step chitosan electrochemical de-
of 0.05 μM under light irradiation. Additionally, this photo- position, we fabricated 3D-G based glucose biosensor through
switchable biosensor was applicable for the detection of glucose attaching GOD/CNTs/Fc-chitosan biocomposite film on the surface
with glucose dehydrogenase (GDH) entrapped in the multilayer of 3D-G electrode (Liu et al., 2014). The prepared enzyme electrode
film. Niu's group constructed a glucose biosensor by the im- presented good performance for the detection of glucose with
mobilization of GOD in CNT-AuNPs-PEI-functionalized ionic liquid high sensitivity, good stability and rapid response.
(PFIL) composite film (Jia et al., 2008). The introduced PFIL acted as
a multifunctional polymer, which dissolved CNTs, protected 4.2. Microfluidic enzymatic biosensors
AuNPs, and retained the bioactivity of GOD.
OMCs with well-ordered pore structure, high specific pore Except for improving the detection sensitivity using carbon
volume and large surface area, are another carbon nanomaterials namomaterials, we also endeavored to develop low-cost and
suitable for the application of electrochemical enzymatic bio- portable microfluidic enzymetic biosensors. Patterned paper-
sensors. The electrochemical behaviors of various electroactive based microfluidic techniques, pioneered by Whitesides' group
compounds were systematically studied at OMCs and CNTs mod- (Martinez et al., 2007), show great promise for designing cheap
ified electrodes (Zhou et al., 2008a). It was found that OMCs-based and disposable analytical devices on account of their facile fabri-
electrode exhibited more favorable electron transfer kinetics than cation, easy-to-use, and ease of scale-up (Cate et al., 2015; Cheng
that at CNTs-based electrode, making OMCs attractive for the et al., 2010; Dungchai et al., 2009; Lou et al., 2013). In 2013, we
electrochemical enzymatic biosensors. Indeed, we discovered that developed self-powered 3D origami microfluidic ECL enzymetic
OMCs-based alcohol dehydrogenase (ADH) and GOD electrodes biosensors for glucose detection (Zhang et al., 2013). In this device,
showed faster response time and higher sensitivity relative to the primary battery (C|FeCl3|NaCl|AlCl3|Al) was employed to pro-
CNTs-based electrodes, which attributed to high density of edge- vide the stable and portable power supply for luminol ECL system.
plane-like defective sites and large surface area of OMCs (Zhou As illustrated in Fig. 5A, two parts of battery cell and sensing cell
et al., 2008b). were assembled in one device through skillful origami technique.
Single-walled carbon nanohorns (SWNHs) with nanoscale When water and ECL solution were added into the battery cell and
graphene tubules and rough external surface are one kind of sensing cell, respectively, the concentration of glucose was probed
86 X. Jia et al. / Biosensors and Bioelectronics 76 (2016) 80–90

area, good electron conductivity and biocompatibility. Recently, we

demonstrated a compartment-less glucose/O2 BFC using OMCs as
substrates for both bioanode and biocathode (Zhou et al., 2009a).
Due to the excellent electrochemical properties of OMCs, OMCs-
based BFC displayed open circuit voltage (0.82 V) and maximum
power output (8.7 μW cm  2), which were both higher than those of
CNTs-based BFC. Employing SWNHs as the support for redox med-
iator and biocatalyst and Pt nanostuctures as cathodic catalyst, we
assembled one-compartment glucose/O2 BFC, which was operated at
the physiological condition with good performance (Wen et al.,
2010). Then we proposed a one-compartment miniature glucose/air
BFC with SWNH-modified carbon fiber microelectrodes (CFME) for
both the bioanode and biocathode (Wen et al., 2011b). GDH and
bilirubin oxidase (BOD) were immobilized on SWNH/CFME as the
anodic and cathodic biocatalyst, respectively. Commercial available
soft drinks were used as fuels to harvest electric power, suggesting
the great potential for the portable microenergy device.
Besides the progress mentioned above, great attention has
been paid to the development of self-powered biosensors based on
BFCs. Compared with other detection techniques, these BFC-based
biosensors exhibit some unique advantages: (1) the self-powered
biosensors are capable for detection without external power
source; (2) using a multimeter as detector, they do not require
complicated instrument; (3) combined with chip technology, they
are promising for economical, sensitive and portable detection
devices.
Fig. 5. Structures and operation mechanisms of of microfluidic enzymatic bio- In 2010, we for the first time proposed a self-powered bio-
sensors. (A) The self-powered 3D origami microfluidic ECL biosensing device. f: sensor based on the inhibitive effect on microchip enzymetic BFC
front sheet; b1: the sheet folded to the back first; b2: the back sheet. (B) The full- (Deng et al., 2010). The integrated BFC on chip was consisted of
featured ECL closed three-channel and double-BPE system. Reprinted with per-
mission from Zhang et al. (2013, 2014).
GDH-bioanode and laccase-biocathode using CNTs as substrate
and electrochemical enhancement. In the presence of cyanide, the
laccase's oxygen reduction ability was inhibited through affecting
by ECL signal. Additionally, our proposed sensing device could be the type 2 (T2) Cu of the laccase active center, thereby leading to a
applied for practical samples with satisfactory results. decreased power output. A linear range from 3.0  10  7 to
A bipolar electrode (BPE) is an electronic conductor, which si- 5.0  10  4 M with a detection limit of 1.0  10  7 M was obtained.
multaneously works as both anode and cathode with wireless The self-powered microchip biosensor was applicable for the de-
contact (Fosdick et al., 2013). Introducing ECL signal at a single BPE tection of cyanide concentration in real sample. Subsequently, we
pole enables simultaneous readout of large arrays of sensing sig- developed a self-powered biosensor for trace Hg2 þ detection
nals with only a battery or a DC power supply (Arora et al., 2001; based on one-compartment miniature alcohol/O2 BFC (Wen et al.,
Wu et al., 2015). Therefore, BPEs with the advantages of low cost, 2011a). The existence of Hg2 þ could affect the bioactivity of ADH-
ease of operation, and simple instrumentation, are promising for bioanode and BOD-biocathode, consequently resulting in the open
cost-effective point-of-care microfluidic analytical devices. In our circuit voltage decrease. The developed self-powered Hg2 þ bio-
work, we constructed a full-featured ECL sensing platform based sensors showed a linear range from 1 to 500 nM with a detection
on the closed three-channel and double-BPE system (Fig. 5B) limit of 1 nM.
(Zhang et al., 2014). This BPE configuration separated the sensing Inspired by the biological principle of feedback modulation, a
part and the reporting part, enabling simultaneous analysis of self-powered biosensor was developed for acetaldehyde detection
reductants and oxidants in a single device, which expanded the based on ethanol/air enzymatic BFC (Zhang et al., 2012). As de-
application range of ECL system. Ascorbic acid, H2O2 and tripro- picted in Fig. 6A, employing CNTs/IL as electrode substrate, bio-
pylamine were detected in the cathodic reservoir, anodic reservoir catalysts ADH and BOD were immobilized on the anode and
and reporting reservoir, respectively, demonstrating the multi- cathode, respectively. The product of ethanol oxidation acet-
functionality of this bipolar system. With this design, we achieved aldehyde suppressed the forward reaction rate of ADH catalysis,
the analysis of blood sugar with good performance. signaled by the power output signal of the BFC. Thanks to the
inherent superiority of the mechanism, the biosensors exhibited
good sensitivity and selectivity, making it feasible for practical
5. Electrochemical biosensors based on BFCs monitoring acetaldehyde content in water resource.
Recently, we proposed the first model of aptamer-controlled
As a green energy technology, BFCs using biomass and enzymes/ BFCs as self-powered and intelligent logic aptasensors (Zhou et al.,
microorganisms as fuel and biocatalysts, respectively, are active at 2010b). As described in Fig. 6B, patterned ITO electrodes were
mild condition such as ambient temperature and near-neutral pH. modified through LBL assembled multilayer consisted of poly
Especially, enzymatic BFCs possess higher current and power density, (diallyldimethylammonium chloride)/CNTs, enzymes (GOD for
and easily realize miniaturization, which are very attractive for im- bioanode, BOD for biocathode), aptamer (TBA and lysozyme-
plantable medical device (Barton et al., 2004; Cracknell et al., 2008). binding aptamer) and BSA. Similar to the principle of EIS apta-
To fabricate effective BFCs, the biocatalyst immobilization technology sensor described above (Zhang et al., 2009), the electrode interface
is crucial, which should retain enzymatic activity and facilitate would be blocked upon addition of thrombin or lysozyme, re-
electron transfer simultaneously. From this perspective, carbon na- sulting in an increased overpotential for glucose oxidation at the
nomaterials are competitive alternatives due to their large surface bioanode or O2 reduction at the biocathode. Accordingly, open-
 X. Jia et al. / Biosensors and Bioelectronics 76 (2016) 80–90 87

electrocatalysis, biocompatibility, and high surface area, are highly

beneficial for improving the sensitivity and stability of electro-
chemical biosensors. In combination with EIS technique and “solid
state probe” technique, we fabricated a series of label-free apta-
sensors and cytosensors, which were adapted to the microfluidic
chip. These label-free biosensors were simple and low-cost while
balancing with the need for general sensing requirements. Mi-
crochip enzymetic biosensors based on patterned-paper and bi-
polar electrodes were favorable for low-cost portable microfluidic
analytical devices. Self-powered biosensors based on BFCs dis-
played simple operation and instrumentation configuration, using
a multimeter as the detector without external power source. They
are promising for economical, sensitive and portable detection
devices, especially for implantable sensors in vivo.
Despite impressive progress has been already made in the field
of electrochemical biosensors, they are still far from real-life ap-
plication, especially regarding the stability and reproducibility.
Several challenges and obstacles remain to be overcome. Firstly,
these electrochemical biosensors are examined in buffer solution
with good performance. The sample matrix effects should be
considered carefully in real sample analysis. For example, bio-
fouling which originates from the nonspecific adsorption of bio-
molecules on the surface of working electrode, interferes with the
performance of these electrochemical biosensors (Fairman et al.,
2013; Jiang et al., 2015). Interference could be minimized or even
eliminated by diluting real samples with buffer, but at the expense
of higher requirement for the sensitivity. Flourishing advance in
nanotechnology continually produces novel functional nanoma-
terials, which is beneficial for improving sensitivity of electro-
chemical biosensors. New bio-nano interaction wolud be dis-
covered, leading to new labeling or conjugating method with
Fig. 6. The schematic representation of electrochemical biosensors based on BFCs.
(A) Sensing principle for the acetaldehyde sensor based on the ethanol/air BFC.
improved performance. As we demonstrated, multifunctional na-
(B) The sensing strategy for logic aptasensors based on the assembled aptamer- nocomposites, which integrate the optical, electronic and mag-
based BFC, and the truth table and the corresponding circuit for a NAND logic gate. netic properties within a single nanoscale complex, provide new
Reprinted with permission from Zhang et al. (2012) and Zhou et al. (2010b). opportunity for more novel electrochemical biosensors with di-
verse functions. Additionally, several factors such as the situation
circuit potential of the BFC was decreased. In the presence of of electrode surface, sensing interface manufacturing process and
thrombin and lysozyme [input (1,1)], the open-circuit potential operator's skill, have an effect on the reproducibility of electro-
was significantly decreased, giving rise to an OFF signal [output 0]. chemical biosensors. In this regard, future development of mi-
When either [input (0,1), (1,0)] or neither analyst [input (0,0)] was crofluidic chip technology might offer a viable avenue with the
existed, the open-circuit potential was still “activated”, producing high throughput, automation and mass-production. Automated
an ON signal [output 1]. The features of the built system corre- analytical microarray platforms employ the robotic liquid handling
sponded to the equivalent circuit of a NAND logic gate. Therefore, via intelligent software, which can minimize these errors and
our proposed self-powered logic aptasensors were capable for thereby improve the reproducibility. Simultaneously, these ana-
judging whether both specific targets existed, which was poten- lytical microarray platforms can achieve high-throughput, rapid
tially promising for designing self-powered medical diagnostics. analysis of many sensing molecules in one test. This area still
Furthermore, we fabricated an IMP-Reset logic-based reusable, needs to be extensively explored further.
self-powered aptasensor (Zhou et al., 2010a) based on our pre- Future clinic medicine and personalized healthcare will require
vious regenerated aptamer design (Du et al., 2008). The designed online monitoring of metabolites, drugs, and proteins in vitro and
self-powered aptasensor could intelligently probe one specific in vivo. It spurs the development of wearable and implantable
target in the absence of the other target in complex physiological electrochemical biosensors, which enable continuous assessment
samples in a single test. Combined the microfluidic chip design, of the important chemical constituents. This requires the minia-
the developed self-powered aptasensor was useful for portable turization and integration of the whole device. In combination
intelligent medical diagnostics device. with the biocomputing systems, intelligent logic devices for
Table 1 summarizes the analytical performances of various medical diagnosis are envisioned, which can serve as bridges to
electrochemical biosensors developed in our laboratory. process information between biological and electronic systems.
Anyhow, these electrochemical biosensors provide new and at-
tractive opportunities to merge the analytical science with recent
6. Conclusions and future perspectives trends in nanotechnology, microfluidic chip, biosensing networks,
telehealth etc.
Our groups have been endeavoring to develop low-cost, por-
table, highly sensitive and selective electrochemical biosensors.
Towards this goal, engineering the bioelectrochemical sensing Acknowledgments
interface is of paramount importance. Functional nanomaterials,
especially carbon nanomaterials such as graphene, CNTs, SWNHs This work is supported by the National Natural Science Foun-
and OMCs with the advantages of good electron conductivity, dation of China with Grants 21190040, 21075116 and 91227114.
 88
Table 1
Analytical performances of electrochemical biosensors.

Strategy Target LOD Linear range References

EIS aptasensors based on duplex-to-complex design Adenosine 10  7 M 10  7–10  2 M B. Li et al. (2007)

EIS aptasensors based on grafting TBA strand onto ABA strand ATP; thrombin 10  8 M; 10  11 M 10  8–10  4 M; 10  11–10  7 M Du et al. (2008)
EIS aptasensors based on AuNPs amplified sandwich structure α-Thrombin 0.02 nM 0.05–18 nM Li et al. (2008)

X. Jia et al. / Biosensors and Bioelectronics 76 (2016) 80–90

EIS aptasensors based on graphene ATP; Hg2 þ 15 nM; 0.5 nM 15 nM–4 μM; 0.5–500 nM Wang et al. (2012)
EIS aptasensors based on graphene α-Thrombin 0.03 nM 0.03–200 nM Wang et al. (2011)
Solid-state DNA sensor based on GSGHs DNA 10 fM 10  11–5  10  7 M Du et al. (2011b)
Solid-state integrated aptasensor based on GSSHs and SMS ATP 0.023 nM 0.05–56.5 nM Guo et al. (2011)
Solid-state aptasensor based on GSSHs D-VP 5 ng mL  1 5 ng mL  1–56.5 mg mL  1 Du et al. (2012)
MECAS chip based on AuNPs ATP; cocaine 0.3 nM; 70 nM 0.5–1066 nM; 0.1–26.3 μM Du et al. (2011a)
MECAS chip using CC measurements Hg2 þ 1 nM 6–1066 nM Chen et al. (2012)
Cytosensor based on ECR HeLa cells 10 cells mL  1 10–106 cells mL  1 Li et al. (2012)
Solid-state Cytosensor based on LBL assembly of SWNTs/Fc-PEI HeLa cells 10 cells mL  1 10
106 cells mL  1 J. Liu et al. (2013)
Solid-state Cytosensor based on graphene/Fe3O4@luminol-AuNPs HeLa cells 8 cells mL  1 20–104 cells mL  1 Gu et al. (2015)
Enzymatic biosensor based on thionine bridged CNTs/AuNPs NADH 0.7 μM 1 μM–3.25 mM Deng et al. (2008)
Enzymatic biosensor based on CNT-AuNPs-PFIL Glucose Not reported 2–12 mM Jia et al. (2008)
Enzymatic biosensor based on OMCs Glucose 156.52 μmol L  1 0.5–15 mmol L  1 Zhou et al. (2008a)
Enzymatic biosensor based on Nafion-SWNHs Glucose 6 μM 0–6 mM Liu et al. (2008)
Enzymatic biosensor based on PSS-SWNHs H2O2 0.5 μM 3–350 μM Liu et al. (2010)
Enzymatic biosensor based on graphene-PFIL Glucose Not reported 2–14 mM Shan et al. (2009)
Enzymatic biosensor based on graphene/AuNPs/CS Glucose 180 μM 2–14 mM Shan et al. (2010b)
Enzymatic biosensor based on IL-graphene/CS NADH; ethanol Not reported; 5 μM 0.25–2 mM; 25–200 μM Shan et al. (2010a)
Enzymatic biosensor based on g-C3N4 Glucose 11 μM 1–12 mM Tian et al. (2013)
Enzymatic biosensor based on CNTs/Fc-CS on 3D-G electrode Glucose 1.2 μM 5.0 μM–19.8 mM Liu et al. (2014)
Self-powered 3D origami microfluidic enzymetic biosensors Glucose 0.1 mM 0.1–3 mM Zhang et al. (2013)
Enzymatic biosensor based on three-channel closed bipolar system Glucose 50 μM 50 μM–5 mM Zhang et al. (2014)
Self-powered biosensor based on microchip BFC Cyanide 1.0  10  7 M 3.0  10  7 –5.0  10  4 M Deng et al. (2010)
Self-powered biosensor based on ethanol/O2 BFC Hg2 þ 1 nM 1–500 nM Wen et al. (2011a)
Self-powered biosensor based on ethanol/air BFC Acetaldehyde 1 μM 5–200 μM Zhang et al. (2012)
Self-powered and intelligent logic aptasensors based on aptamer-controlled BFCs α-Thrombin; Lysozyme Not reported Not reported Zhou et al. (2010b)
 X. Jia et al. / Biosensors and Bioelectronics 76 (2016) 80–90 89

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