Biomaterials 33 (2012) 1024e1031

Contents lists available at SciVerse ScienceDirect

Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Three-dimensional paper-based electrochemiluminescence immunodevice

for multiplexed measurement of biomarkers and point-of-care testing
Lei Ge a, Jixian Yan a, Xianrang Song b, Mei Yan a, Shenguang Ge c, Jinghua Yu a, *
a

School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China
Cancer Research Center, Shandong Tumor Hospital, Jinan 250117, PR China
c
Shandong Provincial Key Laboratory of Fluorine Chemistry and Chemical Materials, University of Jinan, Jinan 250022, PR China
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 28 August 2011
Accepted 24 October 2011
Available online 9 November 2011

In this work, electrochemiluminescence (ECL) immunoassay was introduced into the recently proposed
microuidic paper-based analytical device (mPADs) based on directly screen-printed electrodes on paper
for the very rst time. The screen-printed paper-electrodes will be more important for further development of this paper-based ECL device in simple, low-cost and disposable application than commercialized ones. To further perform high-performance, high-throughput, simple and inexpensive ECL
immunoassay on mPAD for point-of-care testing, a wax-patterned three-dimensional (3D) paper-based
ECL device was demonstrated for the very rst time. In this 3D paper-based ECL device, eight carbon
working electrodes including their conductive pads were screen-printed on a piece of square paper and
shared the same Ag/AgCl reference and carbon counter electrodes on another piece of square paper after
stacking. Using typical tris-(bipyridine)-ruthenium () - tri-n-propylamine ECL system, the application
test of this 3D paper-based ECL device was performed through the diagnosis of four tumor markers in
real clinical serum samples. With the aid of a facile device-holder and a section-switch assembled on the
analyzer, eight working electrodes were sequentially placed into the circuit to trigger the ECL reaction in
the sweeping range from 0.5 to 1.1 V at room temperature. In addition, this 3D paper-based ECL device
can be easily integrated and combined with the recently emerging paper electronics to further develop
simple, sensitive, low-cost, disposable and portable mPAD for point-of-care testing, public health and
environmental monitoring in remote regions, developing or developed countries.
2011 Elsevier Ltd. All rights reserved.

Keywords:
Electrochemiluminescence immunoassay
Lab on paper
Screen-printed electrode
Biomarker
Point-of-care testing

1. Introduction
While the use of paper for biological assays and point-of-care
diagnostics is not a new concept [1], it has never attracted as
much attention as it does now. Whitesides and coworkers recently
introduced a promising concept of using patterned paper substrate
as microuidic platform for multiplex analyte detection [2,3]. They
aim at developing a simple, inexpensive, portable, disposable and
easy-to-use point-of-care platform for developing countries,
resource-limited and remote regions. Microuidic paper-based
analytical devices (mPADs) have gained more and more attention
and great interest during the recent years [4e8]. Much effort has
been directed toward the development of fabrication [3,9e18],
functionalization [19e29] and quantitative methods [27,30e33] for
mPADs. To date, the primary detection method for the qualitative
and quantitative analysis of multiplex analytes on mPADs is

* Corresponding author. Tel.: 86 531 82767161; fax: 86 531 82765969.

E-mail address: ujn.yujh@gmail.com (J. Yu).
0142-9612/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2011.10.065

colorimetric method [2,3,5,9,10,12,13,17,28,33e35] based on visually comparing the color intensity of the reaction spots by naked
eye or camera phones [35] and portable scanners [27]. With the use
of a uidic timer, more accurate glucose levels were determined on
mPADs by colorimetric method [28]. However, specically quantitative analysis is still needed when the level of an analyte in real
complex biological sample is important for simple, rapid, low-cost
point-of-care testing, public health and environmental monitoring.
Recently, there has been an emerging trend of establishing new
analytical methods, such as electrochemical [34,36] and chemiluminescent [32,37] methods, on paper-based analytical device,
which not only retains the simplicity, low cost, portability and
disposability of paper-based analytical devices, but also provides
new opportunities and directions in the development of precise
and sensitive diagnostic devices. Electrochemiluminescence (ECL),
which combines the advantages of chemiluminescence and electrochemistry, continues to impact diverse areas ranging from
chemical analysis to the molecular-level understanding of biological processes [38]. In comparison to the conventional electrochemical and chemiluminescent methods, the ECL assay not only

L. Ge et al. / Biomaterials 33 (2012) 1024e1031

shows high sensitivity and wide dynamic concentration response

range but also is potential- and spatial controlled. Recently, the
establishment of ECL on mPADs [31] based on the integration of
mPADs and commercialized screen-printed electrodes have
substantially increased the scope of options for detections on
mPADs.
Despite their success, there are still pressing needs for mPADs
strategy, especially to separate and enrich analyte from samples
containing highly abundant nonspecic biological matrixes on
mPADs for point-of-care testing. Immunoassay, as an analytical
technique based on the highly specic interaction between antigen
and antibody, is widely applied in clinical chemistry, pharmaceutical analysis, toxicological analysis, bioanalysis, food safety and
environmental analysis due to its high selectivity, rapid detection
and possible analysis of difcult matrices without extensive pretreatment. The paper-based ELISA [33], rstly proposed by Whitesides, has provided an opportunity and protocol for analytical
methods established on mPADs. It is based on the colorimetric
assays on paper microzone plate. However, colorimetric assay is
still not sufciently sensitive or specic for accurate point-of-care
use. In addition, the visual readout is usually limited to a yes/no
answer, this is not adequate when the level of an analyte is very
important. To overcome these challenges, more sensitive and
qualitative methods should be integrated. ECL immunoassay has
become a powerful analytical tool for highly sensitive and specic
detection of clinical samples [38]. It also gives a sensitive, rapid
alternative to other methods as a detection principle in immunoassay for the point-of-care determination of molecules (e.g.,
proteins, hormones, drugs, nucleic acids and environmental
pollutants) [38]. To the best of our knowledge, no reports about
establishing ECL immunoassay on mPADs have been published.
To combine the ECL immunoassay with mPADs for highthroughput, rapid, simple, portable, disposable, low-cost, and
sensitive point-of-care testing, we demonstrated a threedimensional (3D) paper-based ECL device (Scheme 1) based on
wax-patterned technology and screen-printed paper-electrodes in
this work. 3D mPADs [20,21] are particularly useful because they
permit uid movement in the x-, y-, and z-directions, and therefore, they can accommodate more assays on a smaller footprint
than typical 2D, lateral-ow devices. A 3D mPAD can distribute
a sample from a single entry point into hundreds of test regions

Sheet-A

1025

[20]. Wax printing is rapid, inexpensive, insulative and particularly

well-suited for producing large lots (hundreds to thousands) of
prototype mPADs [15e17]. The screen-printed paper-electrodes will
be more important for further development of paper-based ECL
device in low-cost and disposable application than commercialized
ones [30,36,39]. In this 3D paper device, carbon working electrodes
including their conductive pads were screen-printed on a piece of
square paper (named paper-A, 30.0 mm  30.0 mm) and shared the
same Ag/AgCl reference and carbon counter electrodes on another
piece of square paper (named paper-B, 30.0 mm  30.0 mm) after
stacking. The wax patterns around the electrodes on paper-A and
paper-B constituted reservoirs of the electrochemical cells.
The detection of a panel of biomarkers can signicantly improve
the diagnostic value of biomarkers [40]. Thus, as a model, four
tumor markers (r-fetoprotein (AFP), carcinoma antigen 125
(CA125), carcinoma antigen 199 (CA199) and carcinoembryonic
antigen (CEA)), showing considerable signicance in early
screening and clinical diagnosis of some tumor diseases [41], were
used as model analytes. In this work, eight electrodes were screenprinted on paper-A for determination of the four tumor marker
(every two working electrodes for one tumor marker) to obtain
more exact results. Ultimately, with the aids of a simple homemade device-holder, the concentrations of AFP, CA125, CA199 and
CEA in real human serum samples were detected using tris(bipyridine)-ruthenium () (Ru(bpy)2
3 ) - tri-n-propylamine (TPA)
ECL system under the optimum conditions. This work could not
only make contribution to further expand detection mode on
mPADs, but also can be easily integrated and combined with the
recently emerging class of paper electronics [42,43] to further
develop simple, sensitive, low-cost, disposable and portable pointof-care testing device without device-holder in our future work.
2. Materials and methods
2.1. Reagents and materials
Mouse monoclonal capture and Ru(bpy)2
3 -labeled signal AFP, CA125, CA199 and
CEA antibodies were purchased from Linc-Bio Science Co. Ltd. (Shanghai, China).
Chitosan and bovine serum albumin (BSA) was obtained from SigmaeAldrich
Chemical Co. (St. Louis, MO). Glutaraldehyde (GA, 25% aqueous solution) was
purchased from Alfa Aesar China Ltd. Whatman chromatography paper #1
(200.0 mm  200.0 mm) (pure cellulose paper) was obtained from GE Healthcare
Worldwide (Pudong Shanghai, China) and used with further adjustment of size.

A
30 mm

Sheet-B

Screen-printed carbon
working electrodes

4 mm
30 mm
Screen-printed carbon
counter electrode

B
30 mm

7 mm

Screen-printed Ag/Agcl
reference electrode

Scheme 1. The schematic representation of process to fabricate 3D paper-based ECL device. Paper sheets were rstly patterned in bulk using a wax printer. Then electrodes were
screen-printed on sheet-A and sheet-B respectively in bulk. Finally, sheet-A and sheet-B were cut to paper-A and paper-B with the same size (30.0 mm  30.0 mm).

1026

L. Ge et al. / Biomaterials 33 (2012) 1024e1031

Ultrapure water obtained from a Millipore water purication system (g18 MU, MilliQ, Millipore) was used in all assays and solutions. Blocking buffer for blocking the
residual reactive sites on the antibody immobilized paper was pH 7.4 phosphate
buffer solution (PBS) containing 0.5% BSA and 0.5% casein. To minimize unspecic
adsorption, 0.05% Tween-20 was spiked into 10.0 mM pH 7.4 PBS as washing buffer.
Fluorescein isothiocyanate (FITC)-labeled CA125 antibodies and human AFP, CA125,
CA199 and CEA standard solutions (0.5 mg mL1) were from Linc-Bio Science Co. Ltd.
(Shanghai, China). The clinical serum samples were from Shandong Tumor Hospital.
All other reagents were of analytical grade and used as received.
2.2. Fabrication of this 3D paper-based ECL device
Wax was used as the paper hydrophobization and insulation agent in this work.
This 3D paper-based ECL device was comprised of two layers of patterned rectangular papers with the same size (30.0 mm  30.0 mm, named paper-A and paper-B
respectively below). Firstly, as shown in Scheme 1, paper-A and paper-B were
produced in bulk (named sheet-A and sheet-B respectively below). The shape for
patterning this 3D paper-based ECL device was designed using Adobe illustrator CS4.
On paper-A, the wax-patterned contains a circular contacting zone (7.0 mm in
diameter) surrounded by eight working zones (4.0 mm in diameter, 1.0 mm edge-toedge separation) with paper channels to connect them. On paper-B, there is only
a circular contacting zone (7.0 mm in diameter) with the same position as the one on
paper-A. The fabrication process of the wax patterns on sheet-A and sheet-B
includes only two steps after designing on the computer: (1) print the two wax
patterns onto the surface of sheet-A and sheet-B respectively with wax printer
(FUJIXEROX Phaser 8560DN, Japan); (2) bake the wax-printed papers in an oven at
130  C for 150 s to let the printed wax melt and penetrate through the paper to form
the hydrophobic and insulating patterns. These two steps can be nished within
2 min.
The wax-patterned sheet-A and sheet-B were then ready for printing electrodes
on their hydrophilic zones after cooling to room temperature (within 1 min)
(Scheme 1). In addition, due to the small size of this device, the silver wires and
contact pads were unnecessary and can be directly replaced by carbon ink and Ag/
AgCl ink respectively. This will be very important for the further development of this
device in low-cost application. For sheet-A, eight working electrodes containing the
wires and contact pads were screen-printed in the dened area on sheet-A using
carbon ink. The working electrodes were all aligned to the working zones. For sheetB, a counter electrode and a reference electrode were screen-printed in the dened
contacting zone on sheet-B using carbon ink and Ag/AgCl ink respectively. After that,
sheet-A and sheet-B were cut to paper-A and paper-B with the same size. The eight
working electrodes on paper-A will share the same reference and counter electrodes
on paper-B after stacking. The wax patterns around the electrodes constituted
a reservoir of the electrochemical cell with a volume of 35 mL on paper-A and 10 mL
on paper-B. The scanning electron microscopy (SEM) images of this 3D paper-based
ECL device were recorded on a JEOL JSM-5510 scanning electron microscope. The
contact angle tests were performed on contact angle measurement (Model OCA40,
Dataphysics).
In addition, the front and back surfaces of the wax-patterned electrochemical
cell on paper-A and paper-B are open to atmosphere, thus, for paper-A, the working
electrodes can be washed by adding PBS or washing buffer to the back of the circular
contacting zone while bending the paper-A to a inverted-U type as shown in
Fig. S1A. Then a piece of U-type blotting paper was contacted the front of the
working electrodes (Fig. S1C, D). The washing buffer goes through the paper and
migrates along the paper channels by the capillary and gravity action to wash the
working electrodes and carries the unbound reagents with it into the blotting paper.
This washing procedure was repeated twice by changing the bend axis, indicated by
the arrow in Fig. S1A, B, to make sure the washing was performed completely. For
paper-B, the washing procedure can be performed according to method proposed by
Cheng et al. [33]. The printed electrodes will rmly attach to the paper surface due to
the penetration of binding reagents in the inks into the paper matrix. And they will
not break or peel off from the device upon washing and folding [39]. This effective
washing procedure was used in this work consistently and acquiescently. The
washing process was important for preventing the nonspecic binding and
achieving the best possible signal-to-background ratio. Another purpose for this
washing procedure was to stop the incubation reaction at exactly same time.
2.3. Preparation of 3D paper-based ECL immunodevice
As shown in Scheme 2, the 3D paper-based ECL Immunodevice was constructed
by immobilizing the corresponding capture antibodies on the working electrodes
through chitosan coating and GA cross-linking. First, 2.0 mL of 0.5 mg mL1 chitosan
were coated on each working electrode and dried at room temperature. After activating with 2.5% GA (in 50 mM, pH 7.4 PBS) for 1 h and washing with PBS, 2.0 mL of
AFP, CA125, CA199 and CEA antibodies (20 mg mL1) were applied to the corresponding two working electrodes, respectively, and reacted at room temperature for
50 min. Subsequently, excess antibodies were washed with PBS. We then blocked
each working electrodes by adding 2.0 mL of blocking buffer to block possible
remaining active sites against nonspecic adsorption, and allowing the working
electrodes to dry for 15 min under ambient conditions. After another washing with

washing buffer, the resulting 3D paper-based ECL Immunodevice was obtained and
stored at 4  C in a dry environment prior to use. The scanning electron microscopy
(SEM) images of this 3D paper-based ECL immunodevice were recorded on a JEOL
JSM-5510 scanning electron microscope.
2.4. ECL assay procedure of this 3D paper-based ECL immunodevice
The ECL assay procedures on this 3D paper-based ECL device were shown in
Scheme 2, and a detailed procedure was described below. To carry out the immunoreactions and ECL detections, 2.0 mL sample solution containing different
concentration of AFP, CA125, CA199 and CEA in PBS was added to each working
electrode and allowed to incubate for 30 min at room temperature, followed by
washing with washing buffer according to the procedure mentioned above. Then
Ru(bpy)32-labeled signal antibodies (2.0 mL, 10 mg mL1) was added to corresponding working electrodes, and allowed to incubate for 30 min at room
temperature.
After washing with washing buffer again, for ECL assay, the 3D paper-based ECL
immunodevice was integrated with a newly designed device-holder, which was
used to x and connect the 3D ECL device to the ECL system (Scheme 3). This foldertype device-holder was comprised of two simple circuit boards, name board-A and
board-B, with conductive pads on them (Scheme 3a). Firstly, in detail, paper-A was
placed face down onto board-A (Scheme 3b). After that, paper-B was placed face up
onto paper-A (Scheme 3c). Then the device-holder was clamped to make the 3D
paper-based ECL device stacked closely (Scheme 3d). Due to the same size of paperA, paper-B, board-A and Board B, the contacting zones and conductive pads on 3D
paper-based ECL device and device-holder were aligned exactly and also connected
closely with the aids of the pressure. Ultimately, 40 mL of PBS solution containing
0.01 mM TPA was drop to electrochemical cell through the hole on board-B (Scheme
3d) and then the device-holder was placed in front of the photomultipier tube (PMT,
detection range from 300 to 650 nm) biased at 800 V. With the aid of a sectionswitch assembled on an MPI-E multifunctional electrochemical and chemiluminescent analytical system (Xian Remex Analytical Instrument Ltd. Co.), eight
working electrodes were sequentially placed into the circuit to trigger the ECL
reaction in the sweeping range from 0.5 to 1.1 V at room temperature. For reusing
this 3D paper-based ECL device, 50 mL elution buffer were introduced to regenerate
paper-A, and allowed it to dry for 2 min. Then 50 mL of washing buffer was added to
wash paper-A twice. To make sure the regeneration was performed completely, the
regeneration procedure was repeated twice. Paper-B was washed with water for
recycling.

3. Results and discussion

3.1. Characterization of this 3D paper-based ECL immunodevice
This 3D paper-based ECL device was fabricated on pure cellulose
paper. The hydrophilic electrochemical cells were fabricated by
printing wax on the paper surface in a high-resolution printing
mode (Fig. 1A). Owing to the 3D porous structure of paper, the
melted wax can penetrate into the paper to decrease the hydrophilicity of paper remarkably (Fig. 1B, C). The contact angle on the
front and back side wax-penetrated surface is 113 and 108
respectively. After the curing process, the unprinted area maintains
good hydrophilicity, exibility and 3D porous structure and will not
affect the further screen-printing of electrodes.
The surface morphology of the screen-printed working electrodes is an important factor affecting its sensing performance. The
morphologies of chitosan membrane modied and capture antibodies/chitosan membrane modied screen-printed working
electrodes were characterized by SEM. As shown in Fig. 1E, different
from the uniform chitosan lm (Fig. 1D), when the capture antibodies was immobilized on the chitosan membrane modied
screen-printed working electrodes by GA cross-linking, an obvious
aggregation of the trapped biomolecules could be observed on the
electrode surface (Fig. 1E), indicating the successful assembly of the
capture antibodies on the electrode surface [44].
3.2. ECL emission on 3D paper-based ECL immunodevice
Using CA125 as a model, the ECL responses of the carbon/chitosan/GA screen-printed electrode immobilized with sandwich
immunocomplexes labeled with Ru(bpy)2
3 in the presence of TPA
are shown in Fig. 2. One ECL peak appeared at 1.1 V in the anodic

L. Ge et al. / Biomaterials 33 (2012) 1024e1031

1027

CS
1

2
GA

BSA

4
AFP

CA125 CA199

CEA
ECL

Capture Antibodies
Antigens
Ru-labeled
5

Signal Antibodies

Scheme 2. Schematic representation of the fabrication and assay procedure for this 3D paper-based ECL device. 1) screen-printed carbon working electrode; 2) after chitosan
modication; 3) after immobilization of capture antibodies; 4) after blocking and washing; 5) after capturing and washing; 6) after incubation with signal antibodies, washing and
triggering ECL reaction.

process, generated from Ru(bpy)3*2 that is formed by the redox

reaction between TPA* produced during TPA oxidation and
2
Ru(bpy)3
3 produced from the oxidation of Ru(bpy)3 [45]. Clearly,
the sandwich immunocomplexes (Fig. 2B), in which the capture
antibody was immobilized on the surface of electrode and the
signal antibody was tagged with Ru(bpy)2
3 , gives a much higher
ECL response than the nonspecic adsorption of signal antibodies
without antigen (Fig. 2A), indicated very low levels of nonspecic
adsorption of labeled signal antibodies were always observed in
absence of antigen. Furthermore, the ECL intensity increased with
the increasing of the concentration of antigen (Fig. 2C). Therefore, it
could be applied to the sensitive determination of antigens in this
3D paper-based ECL immunodevice.

3.3. Optimization of detection conditions

For paper-based point-of-care testing, the analytical cost,
sensitivity and time efciency is very important. For the analytical
cost, reducing the pipetting volume of solutions into working zones
would decrease the analytical cost remarkably by saving the
reagents. Thus, FITC-labeled CA125 antibodies were used to

Board-B
Conductive pads

Stacking paper-A

Wire
(b)

(a)
Stacking paper-B

Board-A

Adding TPA

(d)

Clamping
(c)

Scheme 3. Schematic representation of the facile home-made device-holder.

Fig. 1. SEM images of A) the boundary of wax pattern: left is pure paper, right is waxprinted paper; B) front face of wax-penetrated paper; C) back face of wax-penetrated
paper; D) chitosan modied screen-printed working electrode; E) antibodies/chitosan
modied screen-printed working electrode.

1028

L. Ge et al. / Biomaterials 33 (2012) 1024e1031

240
a
b
c

ECL intensity (a.u.)

200
160
120
80
40
0
0.5

0.6

0.7
0.8
0.9
Potential (V)

1.0

1.1

Fig. 2. ECL-potential curves of A) background without antigens; B) after a sandwich

immunoreaction with 2.5 U mL1 CA125 and C) 5.0 U mL1 CA125.

investigate the optimum pipetting volume of solutions onto each

working electrode. The investigation procedures included (i)
immobilization of the FITC-labeled CA125 antibodies with different
volumes on the chitosan modied screen-printed working electrode on paper-A; (ii) this screen-printed working electrode was
washed twice by following the procedure mentioned above; (iii)
the uorescence images of the paper channel and the working
electrode were investigated on an inverse uorescence microscope

(ChangFang CFM-500E, China). Fig. 3A showed that when the

pipetting volume was 2.0 mL, the uniformity and sufciency of
antibodies on the working electrode was obtained. Thus, considering the analytical cost, 2.0 mL was selected as the optimal pipetting volume of all reagents. In addition, this small pipetting volume
could further avoid the outow of the solutions from working
electrodes to the paper channels and reduce the cross-talk between
adjacent electrodes to increase the signal-to-background ratio and
the sensitivity of this 3D paper-based ECL immunodevice.
The incubation process was performed at room temperature.
The successful development of the multiplex immunoassay
required that the common incubation time must be suitable for all
analytes. The effects of incubation time on the ECL intensity of this
3D paper-based ECL immunodevice were investigated as follows.
With an increasing incubation time, all the ECL intensities for
25 U mL1 CA125, CA199 and 25 ng mL1 AFP and CEA increased
quickly and have reached their maximum values at 30 min (Fig. 3B),
indicating the maximum formation of these sandwich immunocomplexes. Thus, an incubation time of 30 min was selected in the
further study. As shown in Fig. 3C, the ECL intensity of this 3D
paper-based ECL immunodevice showed great dependence on the
pH value of the solution. The ECL intensity increased from 5.5 to 8.0,
and the maximum was observed at pH 7.4e8.0. Since the optimal
pH value for the biological systems was 7.4, the ECL detection was
performed in pH 7.4 PBS containing 0.01 mM TPA.
3.4. Analytical performance
The analytical performance of this method was veried by
applying 2.0 mL of samples of human AFP, CA125, CA199 and CEA

Fig. 3. Parameters affecting ECL signals in 3D paper-based ECL immunodevice. A) the pipetting volume affects the uniformity and sufciency of protein immobilization (4 mm
diameter paper zones); B) effects of incubation time on ECL intensities at 25 U mL1 CA125, CA199 and 25 ng mL1 AFP, CEA concentration, where n 11 for each point; C) effect of
pH value on ECL intensities with 25 U mL1 CA125 as a model.

L. Ge et al. / Biomaterials 33 (2012) 1024e1031

standard solutions at various concentrations in PBS under the

optimum conditions. The ECL response and calibration curves for
the four tumor markers were shown in Fig. 4. With the increasing
concentrations of AFP, CA125, CA199, and CEA, good correlations
between the concentration of tumor markers and the ECL peak
intensity with a similar wide dynamic range (0.5e100 ng mL1,
1.0e100 U mL1, 0.5e100 U mL1, and 1.0e100 ng mL1) that
covered most of the levels in human plasmas and serums was also
observed (Fig. 4), respectively. The linear regression equations were
I 13.7[AFP](ng$mL1) 165.9 (R 0.9978), I 19.8[CA125]
(U$mL1) 129.9 (R 0.9971), I 12.6[CA199] (U$mL1) 175.7
(R 0.9968) and I 18.2[CEA] (ng$mL1) 156.0 (R 0.9963),
respectively, where I was the ECL intensity. The limits of detection
at a signal-to-noise ratio of 3 for four tumor markers were
0.15 ng mL1, 0.6 U mL1, 0.17 U mL1, and 0.5 ng mL1, respectively.
Thus, on the basis of this standard curve, the ECL approach should
be useful for the determination of the four tumor markers in real
serum samples, due to the cutoff values of the four tumor markers
in clinical diagnosis are 25 ng mL1, 35 U mL1, 35 U mL1, and
5 ng mL1, respectively [41].
To verify the applicability and validity of the present method to
real biological samples, the determination of AFP, CA125, CA199
and CEA in real human serum was conducted by this method and
compared with those obtained by the commercially used electrochemiluminescence method in Cancer Research Center of Shandong Tumor Hospital. The results were shown in Table 1, the

1000

An excellent ECL immuno-array must exclude cross-talk, which

generally results from the diffusion of signal reporter from one
electrode to neighboring electrodes. In this work, the Ru(bpy)2
3 labeled antibodies was used as the ECL signal reporter. According to
the design of the electrochemical cell in paper-A (Scheme 1), the
diffusion of signal reporter from one electrode to neighboring
electrodes should go through two long paper channels, and the
washing direction could effectively exclude this diffusion. Hence,
the possible cross-talk could be avoided.
To further conrm the resistance to cross-talk, the crossreactivity between analytes and non-cognate antibodies was also
investigated. In this 3D paper-based ECL immunodevice, four
different capture antibodies for AFP, CA125, CA199, and CEA were
immobilized on the eight working electrodes separately. The crossreactivity was evaluated by comparing the ECL intensities of this 3D
paper-based ECL device incubated with blank solution, 50 ng mL1

2500

1500

50.0

800

20.0
5.0
0.5 1.0

400
0

AFP (ng.mL )

800

100.0

2000

75.0

ECL Intensity (a.u.)

ECL Intensity (a.u.)

1200

3.5. Evaluation of cross-talk on this 3D paper-based ECL device

75.0

1200

1400

agreement between the two methods was acceptable. According to

the criteria suggested by Li [41] the blood donor of sample-1 may
be at the highest liver cancer risk; the blood donor of sample-2 may
be placed in the class of the highest ovarian cancer risk; the blood
donor of sample-3 can be placed in the class of the highest
pancreatic cancer risk and the blood donor of sample-4 can be at
the highest colorectal cancer risk.

100.0

1600

1600

1029

600
400
200

2000
1500

60.0

1000
500
1.0 3.0

15.0
5.0

0
CA125 (U.mL )

1000
500
0

0
0

20
40
60
80
AFP Concentration (ng.mL )

1500

2500

50.0
25.0

500
5.0
0.5 1.0

1000
0

CA199 (U.mL )

800

20
40
60
80
CA125 Concentration (U.mL )

600
400
200

100

100.0

2000

75.0
50.0

1500

70.0

1000

ECL Intensity (a.u.)

ECL Intensity (a.u.)

1200

100.0

1600
1400

100

2000
1000

30.0

1500

500

5.0
1.0 3.0

0
CEA (ng.mL )

1000
500
0

0
0

20
40
60
80
CA199 Concentration (U.mL )

100

20
40
60
80
CEA Concentration (ng.mL )

Fig. 4. Calibration curves for AFP, CA125, CA199 and CEA (eleven measurements for each point). Inset: ECL emission intensities.

100

1030

L. Ge et al. / Biomaterials 33 (2012) 1024e1031

Table 1
Assay results of real human serum by the proposed and reference method.
Samples

AFP concentration (ng mL1)

Proposed method

Reference method

Relative error (%)

Proposed method

Reference method

Relative error (%)

Sample1
Sample2
Sample3
Sample4

60.9  2.2
16.1  0.3
7.9  0.2
20.7  1.0

63.2  2.7
15.4  0.6
7.7  0.3
22.1  0.9

3.5
4.9
4.3
6.4

6.2  0.3
75.1  2.4
13.8  0.7
19.1  1.1

6.7  0.4
73.8  3.4
15.2  0.9
19.6  1.3

7.6
1.7
9.2
2.8

Samples

CA199 concentration (U$mL1)

Proposed method
Reference method
36.2  1.6
33.7  1.3
23.9  1.1
25.4  1.1
93.8  3.3
87.6  3.7
52.6  2.0
49.4  2.4

Relative error (%)

7.5
5.9
7.1
6.4

CEA concentration (ng mL1)

Proposed method
Reference method
41.2  2.0
40.2  2.1
37.9  1.7
38.6  2.3
26.5  1.2
27.9  1.4
54.1  2.2
55.3  2.4

Sample1
Sample2
Sample3
Sample4

CA125 concentration (U$mL1)

Relative error (%)

2.5
3.9
4.7
2.1

* Average of eleven measurements.

AFP, 50 U mL1 CA125, 50 U mL1 CA199 or 50 ng mL1 CEA. As

expected, only the working electrodes prepared with corresponding capture antibodies showed obvious ECL responses (Fig. 5).
Obviously, the cross-reactivity between analytes and non-cognate
antibodies was negligible. On the other hand, as shown in Fig. 2A,
the low nonspecic adsorption of the signal reporter on working
electrodes indicated the potential cross-talk between the electrodes could be completely avoided in this 3D paper-based ECL
device. Thus, simultaneous multianalyte immunoassay could be
performed on this designed disposable 3D paper-based ECL device.
3.6. Reproducibility and precision of this 3D paper-based ECL
immunodevice
This 3D paper-based ECL immunodevice could be regenerated,
due to the stably covalent immobilization of antibodies on electrodes surface, for reuse by a simple and effective procedure with
the elution reagents, which is very important for the further
development of mPADs in low-cost application. Furthermore, the
regeneration must avoid being harmful to the activity of the
immobilized antibodies and damaging the bonds between the
antibodies and electrodes surface when the dissociation of the
immunocomplexes occurs [46]. Different elution reagents were
tested using 50 U mL1 CA125, CA199 and 50 ng mL1 AFP, CEA for
regeneration purposes, such as salt solution of organic solvent, high
concentration, buffer with low pH value, and diluted alkali solution

of different concentrations and pH, were tested. The regeneration

efciencies (REs), proposed by Yakovleva et al. [47], were calculated
according to the following equation.


RE

1


RT  B
 100%
T

(1)

Where, RT represents the ECL intensity obtained after the regeneration cycle, B is the ECL intensity for blank, and T is the ECL
intensity before applying any regeneration step. 0.1 M glycine-HCl
(pH 2.1) showed the best regeneration efciency at more than
97% for these four tumor markers, which was chosen as the elution
reagent for the regeneration of this 3D paper-based ECL immunodevice. With the regeneration procedure, this 3D paper-based ECL
immunodevice could be used for 25 cycles with an acceptable
reproducibility. In addition, bulk and frequent operations of the
immunoreactions on working electrodes on paper-A can be realized
without considering the its inuence and contaminate to reference
and counter electrode on paper-B, this could further decrease the
analysis cost due to the increased service life of the electrodes,
especially the expensive Ag/AgCl reference electrode, on paper-B;
When this 3D paper-based ECL immunodevice was stored dry at
4  C (sealed) and measured at intervals of 3 days, No obvious
change was observed after storing for 3 weeks, indicating that this
3D paper-based ECL immunodevice was stable for storage or longdistance transport in remote regions and developing countries.

4. Conclusions

Blank
AFP
CA125
CA199
CEA

1200

ECL Intensity (a.u.)

1000
800
600
400
200
0

Fig. 5. ECL response for different antigens on different electrodes. A) AFP antibodies/
chitosan modied working electrodes; B) CA125 antibodies/chitosan modied working
electrodes; C) CA199 antibodies/chitosan modied working electrodes; D) CEA antibodies/chitosan modied working electrodes.

In this work, a 3D paper-based ECL immunodevice was demonstrated for the rst time to perform high-performance and multiplex
point-of-care diagnostics with simple operation. This proposed 3D
paper-based ECL immunodevice has combined the simplicity and
low-cost of mPADs and the sensitivity and specicity of ECL immunoassay. The advantages of this conguration includes: (1) the
separated paper-electrodes system will be very benecial to the high
integration and various distributional pattern of working electrodes
on paper without considering the position and pattern of reference
and counter electrode; (2) Bulk operations of the immunoreactions
on working electrodes can be realized without considering the its
inuence and contaminate to reference and counter electrode; (3) It
will be easily understandable to obtain the ECL response just
through stacking two papers in a simple home-made device-holder.
This proposed 3D paper-based ECL immunodevice, with highthroughput, rapid, sensitive, stable and reusable ECL response to
trace amount of analyte in real biological samples, will be very useful
when the level of an analyte in real complex biological sample is very
important for simple, rapid, low-cost point-of-care testing in remote

L. Ge et al. / Biomaterials 33 (2012) 1024e1031

regions, developing or developed countries. In addition, this 3D

paper device can be easily integrated and combined with the
recently emerging paper electronics to further develop simple,
sensitive, low-cost, disposable and portable point-of-care testing
device without device-holder in our future work.
Acknowledgements
This work was nancially supported by National Natural Science
Foundation of Peoples Republic of China (No. 21175058); Natural
Science Foundation for Young Scientists of China (Grant No.
51003039).
Appendix. Supplementary material
Supplementary material associated with this article can be found,
in the online version, at doi:10.1016/j.biomaterials.2011.10.065.
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