Content uploaded by Chongwen Wang
Author content
All content in this area was uploaded by Chongwen Wang on Nov 25, 2023
Content may be subject to copyright.
Journal of Hazardous Materials 459 (2023) 132192
Available online 29 July 2023
0304-3894/© 2023 Elsevier B.V. All rights reserved.
Graphene oxide-based colorimetric/uorescence dual-mode
immunochromatography assay for simultaneous ultrasensitive detection of
respiratory virus and bacteria in complex samples
Xiaodan Cheng
a
,
b
, Xingsheng Yang
a
,
b
, Zhijie Tu
a
,
b
, Zhen Rong
a
,
b
,
*
, Chongwen Wang
b
,
c
,
**
,
Shengqi Wang
a
,
b
,
*
a
Bioinformatics Center of AMMS, Beijing 100850, PR China
b
Beijing Key Laboratory of New Molecular Diagnosis Technologies for Infectious Diseases, Beijing 100850, PR China
c
Department of Clinical Laboratory Medicine, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University,
Guangzhou, Guangdong 510000, PR China
HIGHLIGHTS GRAPHICAL ABSTRACT
•The rst 2D colorimetric/uorescence
dual-signal tag used for ICA method.
•Sensitivity improved by 10 and 500
times in colorimetric mode and uores-
cence mode than AuNPs-ICA,
respectively.
•Respiratory virus and bacteria can be
simultaneously detected within 20 min
through one ICA strip.
•Simultaneous detection of H1N1 and
S. pneumoniae was rstly achieved by
ICA method.
ARTICLE INFO
Editor: Lingxin Chen
Keywords:
Inuenza A
Streptococcus pneumoniae
Film-like dual-signal nanotag
Colorimetric/uorescence dual mode
Immunochromatography assay
ABSTRACT
A point-of-care testing biosensor that supports direct, sensitive, and simultaneous identication of bacteria and
virus is still lacking. In this study, an ultrasensitive immunochromatography assay (ICA) with colorimetric/
uorescence dual-signal output was proposed for exible and accurate detection of respiratory virus and bacteria
in complex samples. Colorimetric AuNPs of 16 nm and two layers of quantum dots (QDs) were coated onto the
surface of monolayer graphene oxide (GO) layer by layer to form a multilayered dual-signal nanolm. This
material not only can generate strong colorimetric and fluorescence signals for ICA analysis but also can provide
larger surface area, better stability, and superior dispersibility than conventional spherical nanomaterials. Two
test lines were built onto the ICA strip to simultaneously detect common respiratory virus inuenza A and
respiratory bacteria Streptococcus pneumoniae. The dual-signal mode of assay greatly broadened the applied range
of ICA method, in which the colorimetric mode allows for quick determination of virus/bacteria and the uo-
rescence mode ensures the highly sensitive and quantitative detection of target pathogens with detection limits
* Corresponding authors at: Bioinformatics Center of AMMS, Beijing 100850, PR China.
** Corresponding author at: Beijing Key Laboratory of New Molecular Diagnosis Technologies for Infectious Diseases, Beijing 100850, PR China.
E-mail addresses: rongzhen0525@sina.com (Z. Rong), wangchongwen1987@126.com (C. Wang), sqwang@bmi.ac.cn (S. Wang).
Contents lists available at ScienceDirect
Journal of Hazardous Materials
journal homepage: www.elsevier.com/locate/jhazmat
https://doi.org/10.1016/j.jhazmat.2023.132192
Received 22 May 2023; Received in revised form 20 July 2023; Accepted 29 July 2023
Journal of Hazardous Materials 459 (2023) 132192
2
down to 891 copies/mL and 17 cells/mL, respectively. The proposed dual-mode ICA can also be applied directly
for real biological and environment samples, which suggests its great potential for eld application.
1. Introduction
Acute respiratory infections (ARIs), which are caused by a wide
range of respiratory viruses and bacteria, are a worldwide health
problem with high infectivity and morbidity [1]. The World Health
Organization declared that ARIs bring more than three million deaths
worldwide each year [2]. The respiratory pathogenic microorganisms
are highly resistant to the environment and can spread rapidly through
close person-to-person contact or environmental media (e.g., air, water,
and object surface) [3]. The infection symptoms (e.g., dyspnea, fever,
and cough) and epidemic characteristics of common respiratory patho-
gens (viruses and bacteria) are also similar [4,5]. Thus, accurately
determining the pathogen species by clinical symptoms is difcult [6,7].
Given that bacterial and viral infections are treated differently, early and
accurate determination of causal agent is the key to minimizing losses
and saving lives.
Polymerase chain reaction (PCR) and genetic sequencing are the
main laboratory testing techniques for pathogenic microorganisms, and
they can provide accurate results of genetic information of the targets
[8–10]. However, one nucleic acid detection system is difcult for uni-
versal detection of pathogens due to the difference in biological struc-
ture between bacteria and viruses [11]. For example, bacteria usually
need to be lysed before detection to obtain template DNA for PCR pro-
cedure, whereas most viruses require to undergo reverse transcription
[12]. The primers and systems of genetic sequencing for bacteria and
viruses are also different [13]. In addition, the current nucleic acid tests
are laborious (require multiple steps), expensive (require sophisticated
instruments), and time consuming (generally need 2–6 h) [14]. There-
fore, a simple method for rapid, direct, sensitive, and simultaneous
detection of respiratory viruses and bacteria is urgently needed.
Nowadays, various nanomaterial-guided point-of-care testing
(POCT) biosensors have been developed for rapid and sensitive detec-
tion of biological molecules [15–17]. Given its unique advantages of
rapidity, simplicity, high specicity, and low cost, immunochromatog-
raphy assay (ICA) has been considered as one of the most feasible POCT
methods and extensively applied in the eld of food safety control,
disease screening, and environmental monitoring [18,19]. However, on
the actual need for pathogen detection, the traditional ICA methods are
still faced with the difculty of insufcient sensitivity and poor quan-
titative capacity [20,21]. The reason is that they adopt colloidal AuNPs
to output visual colorimetric signals. In recent years, many kinds of
novel signal nanomaterials, including uorescence tags, chem-
iluminescence microspheres, surface-enhanced Raman scattering
(SERS) nanotags, and magnetic particles, have been introduced into ICA
system to provide more sensitive and readable signals (e.g., uorescence
signal, chemiluminescence signal, SERS signal, magnetic signal) for
quantication of target [22–26]. However, these novel signal-based ICA
biosensors generally no longer have colorimetric ability to support vi-
sual determination. They also require an additional equipment to mea-
sure the quantiable signal, which are high cost and not portable and are
unsuitable for real eld testing.
In last few years, a class of dual-signal output ICA technologies, such
as colorimetric/uorescence dual-mode ICA, colorimetric/SERS dual-
readout ICA, and colorimetric/photothermal dual-signal ICA, have
been proposed to offer visible and quantiable signals to increase ex-
ibility and performance on eld testing [27–30]. For instance, Hu’s
group and our group respectively conrmed that introduction of
SiO
2
/Au/quantum dot (QD) dual-signal tags into ICA can achieve rapid
screening (colorimetry) and accurate detection (uorometry) of cystatin
C and SARS-CoV-2-specic IgM/IgG in a short time (15–20 min) [31,
32]. Further considering the colorimetric/uorescence dual-mode ICA
only requires a small UV light or uorescence reader to generate more
sensitive uorescence signals. Thus, this combination is more suitable
for on-site use. However, the general performance (sensitivity, versa-
tility, stability, and application range) of current color-
imetric/uorescence dual-readout ICA methods should be further
enhanced for accurate detection of pathogens in complex samples.
Graphene oxide (GO) is a widely used 2D nanomembrane due to its
excellent exibility and stability, good electronic conductivity, and huge
surface area, as well as abundant surface groups (e.g., carboxyl, epoxy,
and hydroxyl), which can act as a multifunctional supporter to integrate
with other nanomaterials to improve the performance of biosensors
[33–35]. Our recent work found that QD-loaded monolayer GO nano-
sheet is a exible lm-like fluorescent nanotag with 2D structure, high
photostability, and strong luminescence and is suitable for sensitive
detection of bacteria on ICA strip [36,37]. Based on these foundations,
we fabricated a novel lm-like colorimetric/uorescence dual-signal
nanotag (GO-Au/QD-QD) by integrating one layer of
density-controlled AuNPs and two layers of QDs onto GO surface. Then,
we introduced it into ICA system for simultaneous detection of the most
common respiratory virus inuenza A (H1N1) and respiratory bacteria
Streptococcus pneumoniae (S. pneumoniae). Compared with the previously
reported ICA methods, the GO-Au/QD-QD-based dual-mode ICA pos-
sesses three obvious innovations. First, the GO-Au/QD-QD nanolm is
the rst 2D colorimetric/uorescence dual-signal tag applied in ICA
method, and this tag can provide enhanced colorimetric/uorescence
signals, larger surface area, better stability, and superior uidity than
common spherical tags. Second, the dual-signal GO-Au/QD-QD inte-
grated into ICA method greatly improved the detection range and
sensitivity of the test strip for pathogenic microorganisms. The colori-
metric mode of the proposed assay can support the rapid screening of
target pathogens without any special apparatus and its uorescence
mode allows the ultrasensitive, quantitative, and real-time detection for
complex samples. Impressively, the limits of detection (LODs) of the
proposed assay using colorimetric and uorescence modes were 10 and
500 times higher than that of traditional AuNPs-based ICA method,
respectively. Third, the dual-mode ICA can simultaneously and sensi-
tively detect respiratory virus and bacteria in complex samples within
20 min. The specicity, accuracy, stability, and practical detection
capability of the proposed method in real environment samples and
biological samples were systematacially demonstrated. To the best of
our knowledge, this work is the rst report on the use of color-
imetric/uorescence dual-signal out ICA method for multiplex detection
of pathogenic microorganisms and we are the rst to achieve the
simultaneous detection of respiratory bacteria and virus by ICA strip. We
believe that the dual-mode GO-Au/QD-QD-ICA has great potential for
on-site monitoring of pathogenic microorganisms.
2. Experimental section
The detailed information of materials, reagents, and instruments
used in the study is provided in Supporting information S1.1.
2.1. Preparation of AuNPs with diameters of 16 and 40 nm
The AuNPs of 16 and 40 nm were both synthesized via a classical TSC
reduction method. Briey, 1 mL of 1 % (w/w) HAuCl
4
solution was
mixed with 98 mL of deionized water, and the mixture was heated. After
boiling, 4 mL of 1 % (w/w) TSC solution was rapidly added to form 16
nm AuNPs, whereas 1 mL of 1 % (w/w) TSC solution was rapidly added
to form 40 nm AuNPs. The solutions with AuNPs of 16 and 40 nm were
held at 100 ◦C with magnetic stirring for 15 min. Finally, the AuNPs
X. Cheng et al.
Journal of Hazardous Materials 459 (2023) 132192
3
suspensions were naturally cooled to room temperature and then stored
at a cool place away from light.
2.2. Preparation of dual-signal GO-Au/QD-QD nanoakes
First, 400 mL of GO solution (2 mg/mL) was added into 1200 mL of
deionized water. The mixture was then sonicated for 15 min to disperse
completely. Second, the GO nanosheets were mixed with 200 mL of PEI
solution (1 mg/mL). The mixture was vigorously sonicated for 30 min.
During this process, both sides of the negatively charged GO sheet were
rapidly coated by the positively charged PEI. The formed GO-PEI
nanosheets were collected by centrifugation (10,000 rpm, 10 min),
washed twice with deionized water, and resuspended in 400 mL of
deionized water. Third, the collected GO-PEI nanoakes were quickly
added to the solution of 40 mL 16 nm AuNPs suspensions. Then, the
mixture was subjected to intense sonication for 30 min to form GO-Au
nanoakes. The GO-Au nanosheets were collected by centrifugation
(7500 rpm, 6 min), washed once with deionized water, and resuspended
in 40 mL of deionized water. Fourth, 80
μ
L of CdSe@ZnS-COOH QDs
(100 nM) was added into the solution. The mixture was vigorously
sonicated for 60 min. During the process, the QDs absorbed on the PEI
sites of the GO-Au nanosheets and distributed among the uniform
AuNPs. The GO-Au/QD nanosheets were collected by centrifugation
(7000 rpm, 6 min). Fifth, the GO-Au/QD nanosheets were reacted with
PEI and CdSe@ZnS-COOH QDs in turn to obtain a higher uorescence
intensity. Thus, the GO-Au/QD-QD nanocomposites were prepared by
PEI-mediated layer-by-layer assembly. Finally, the formed GO-Au/QD-
QD nanosheets were separated by centrifugation (6000 rpm, 6 min)
and resuspended in 20 mL of ethanol for future use.
2.3. Preparation of immuno-GO-Au/QD-QD nanotags
The anti-H1N1 and anti-S. pneumoniae antibodies were conjugated
with GO-Au/QD-QD via EDC/NHS chemistry. Briey, 0.5 mL of GO-Au/
QD-QD nanoakes was centrifuged, resuspended in 500
μ
L of MES
buffer (10 mM, pH 5.6), and mixed with 50
μ
L of EDC (10 mM) and 10
μ
L
of sulfo-NHS (100 mM) under sonication. After activation for 15 min, the
mixture was centrifuged to remove the excess activators (EDC/sulfo-
NHS) and resuspended in 400
μ
L of PBS solution (10 mM, pH 7.4). Then,
12
μ
g anti-H1N1/S. pneumoniae antibodies were added to incubate with
the activated GO-Au/QD-QD solution for 2 h, followed by surface
blocking with 100
μ
L of BSA solution (100 mg/mL) for another 50 min.
The resulting immune-GO-Au/QD-QD nanotags were centrifuged,
washed once with PBST solution (10 mM PBS containing 0.05 % Tween
20), and freeze-dried via a vacuum dryer. Finally, 1 mg of immune-GO-
Au/QD-QD powder was resuspended in 0.1 mL of storage solution [10
mM PBS containing 1 mg BSA (w/v), 0.5 % sucrose (w/v), 0.02 % NaN
3
(w/v)] for subsequent use.
2.4. Multiplex ICA strip preparation for simultaneous detection of H1N1
and S. pneumoniae
For simultaneous detection of H1N1 and S. pneumoniae, the ICA strip
was designed to consist of three parts: a sample pad for specimen solu-
tion loading, an NC membrane with one virus test line and one bacteria
test line, and an absorption pad to generate capillary action. The NC
membrane was pasted on the plastic backing card. Then, the H1N1
antibody (1 mg/mL) and S. pneumoniae antibody (0.8 mg/mL) were
separately prayed onto T1 and T2 lines to capture the immune-GO-Au/
QD-QD-H1N1/S. pneumoniae complexes. A mixture of 50 % goat anti-
mouse IgG (0.8 mg/mL) and 50 % goat anti-rabbit IgG (0.8 mg/mL)
was sprayed onto the control zone (C line) to catch excess immuno-GO-
Au/QD-QD nanotags. After the corresponding antibodies were sprayed,
the NC membrane was dried at 37 ◦C for 3 h. The sample pad and ab-
sorption pad were then assembled on the plastic backing card and
overlapped with the NC membrane by 2 mm to ensure the smooth
movement of the sample solution on the strip. Finally, the fully assem-
bled card was cut into individual ICA strips with a width of 3 mm for
further use.
2.5. Simultaneous detection of H1N1 and S. pneumoniae by dual-mode
GO-Au/QD-QD-ICA
The procedure of detecting H1N1 and S. pneumoniae through the
dual-mode GO-Au/QD-QD-ICA was performed in a 2 mL EP tube. First,
4
μ
L of liquid immuno-GO-Au/QD-QD nanotags and 10
μ
L of 10 ×
running buffer (100 mM PBS solution containing 40 % FBS and 10 %
Tween 20) were added into 90
μ
L of each specimen. The mixture was
vigorously vortexed for 10 s. Then, the ICA strips were inserted into each
tube to start the detection process. After reacting for 20 min, the
colorimetric signal on the C line and two T lines was observed with the
naked eye, whereas the uorescence signal was observed using a UV
light. The quantitative analysis of uorescence signal of the two T lines
was conducted simultaneously via a uorescence strip reader at an
excitation of 365 nm.
2.6. Determination in complex samples
The applicability of the proposed biosensor in complex samples is
also an important consideration. Herein, 12 environmental samples (6
lake water samples and 6 object surfaces samples) and 6 biological
samples were used. The lake water samples were collected from Beijing
Yuyuantan Park. The object surface samples were collected using med-
ical sterile cotton swabs, which were randomly wiped on the hospital
door handles, tables, and chairs and immediately inserted into sterile
tubes with 1 ×running buffer (1000
μ
L) for later use. The sputum
samples were collected from healthy volunteers in a sterile tube and
mixed with 1 mL of 2 ×running buffer. These complex samples were
spiked with certain concentrations of H1N1 and S. pneumoniae (H1N1
with concentrations of 1 ×10
7
, 1 ×10
6
, 1 ×10
5
, and 1 ×10
4
copies/
mL; S. pneumoniae with concentrations of 1 ×10
5
, 1 ×10
4
, 1 ×10
3
,
and 1 ×10
2
cells/mL). Next, the detection procedure was conducted
through the dual-mode GO-Au/QD-QD-ICA method. The procedure was
performed thrice for each sample, and the uorescence data were
collected and averaged before use. Finally, the recoveries (%) of H1N1
and S. pneumoniae were calculated according to the established cali-
bration curves.
3. Results and discussion
3.1. Principle of dual-signal nanoake-based ICA for simultaneous
detection of H1N1 and S. pneumoniae
Scheme 1a exhibits the synthetic process of the dual-signal GO-Au/
QD-QD nanosheet. First, we prepared monodisperse 2D GO nanosheets
as a exible lm-like substrate to provide large surface area for signal
nanoparticle loading. Second, the electronegative AuNPs of 16 nm were
equably absorbed on the negatively charged GO sheet through the
cationic polymer PEI to provide strong colorimetric signal (purple
color). Third, negatively charged QDs were used to ll the vacant sites of
GO-Au nanoake for providing strong uorescence intensity. Finally,
second layer of PEI was introduced onto GO-Au/QD to adsorb the second
layer of QDs for generating enhanced uorescence signals. Through this
strategy, the prepared GO-Au/QD-QD nanoake combines the excellent
dispersibility and exibility of GO nanosheet, the strong colorimetric
properties of AuNPs, and the outstanding uorescence property of
multilayered QDs. Thus, it can act as lm-like dual-signal nanotags for
ICA application.
Scheme 1b and 1c illustrates the preparation of immuno-nanoake
tags and the principle of colorimetric/uorescence dual-signal output
ICA biosensor for on-site and simultaneous detection of respiratory virus
and bacteria, respectively. By virtue of the abundant carboxy groups of
X. Cheng et al.
Journal of Hazardous Materials 459 (2023) 132192
4
QD shell, anti-H1N1/S. pneumoniae antibodies can be efciently conju-
gated with GO-Au/QD-QD nanoakes via EDC/NHS chemistry (Scheme
1b). For ICA detection, the prepared GO-Au/QD-QD immunoprobes
were directly mixed with the target sample and dropped onto the sample
pad of paper strip. Typically, if the sample contained target virus or
bacteria, then the formed GO-Au/QD-QD-H1N1 or GO-Au/QD-QD-
S. pneumoniae complexes migrated along the strip from the sample pad
toward the absorbent pad by capillary forces and then were captured by
pre-coated H1N1/S. pneumoniae antibodies on the T lines. The colori-
metric/uorescence signals of T1/T2 zones became stronger with the
increasing concentrations of H1N1 and S. pneumoniae in the tested
samples. On this basis, the colorimetric mode allows the quick deter-
mination of virus/bacteria and the uorescence mode ensures the highly
sensitive and quantitative detection of target pathogens. The control (C)
line was loaded with a mixture of anti-rabbit and anti-mouse IgG, which
was used to catch the superuous H1N1/S. pneumoniae immunoprobes.
3.2. Characterization of GO-Au/QD-QD nanoakes
The structure and chemical properties of the synthesized GO-Au/QD-
QD nanoakes were systematically characterized. First, transmission
electron microscopy (TEM) was used to characterize the morphology of
the GO-Au/QD-QD nanoakes in various stages. Fig. 1a–d shows the
TEM images of GO, GO-Au, GO-Au/QD, and GO-Au/QD-QD nanosheet,
respectively. As displayed in Fig. 1a, the bare GO sheet is a nearly
transparent membrane with exible structure. Our previous works
veried that electropositive PEI can self-assemble on negatively charged
nanomaterials (e.g., SiO
2
, Fe
3
O
4
, and GO nanosheet) to transform their
surface electrical properties. Fig. S1 shows that the zeta potential of GO
nanosheet increased from −36.9 to 22.5 mV after PEI modication,
which indicates that the PEI-coated GO (GO-PEI) was successfully pre-
pared. Under the drive of sonication, negatively charged AuNPs were
rmly adsorbed onto the surface of GO-PEI through electrostatic in-
teractions, which yielded the GO-Au sheet. Fig. 1b shows that AuNPs of
16 nm were evenly distributed on the surface of GO-PEI nanosheet.
Furthermore, density functional theory (DFT) calculation are under-
taken to shedlight on mechanism of enhanced adsorption capacity of
PEI-coated GO for Au. As is shown in Fig. S2, both structures are in a
stable state after structural optimization. The binding energies of Au
with GO and GO-PEI nanosheet are −2.75 ev and −4.65 ev, respectively.
It representes that PEI acctually have enhanced the binding ability of GO
for Au. From Fig. S2(a)-(b), –COOH groups on GO acts active sites to
adsorb Au, and –COOH stalizing Au has been widely reported [38,39].
Moreover, from Fig. S2(c) the PEI-coated GO could provide more active
sites including the -NH
2
groups to adsorb Au. It has been accepted that
-NH
2
can also combine with Au to generate a weak covalent bond [40].
Besides, Au atoms are simutanueously adsorbed on the surface of PEI in
parallelly. Consequently, PEI-coated GO can stablize Au easily than GO
alone. The remaining sites of the PEI layer could continue to absorb
carboxylated QDs, which resulted in GO-Au/QD nanosheet with a mixed
layer of AuNPs and QDs (Fig. 1c). The formed GO-Au/QD was repeat-
edly reacted with PEI and QDs to fabricate the second layer with
QDs-coated nanosheet (GO-Au/QD-QD). As shown in Fig. 1d, more QDs
gathered on the surface of GO-Au/QD-QD after the absorption of the
Scheme 1. (a) Fabrication procedure of dual-signal GO-Au/QD-QD nanoakes, (b) Preparation of immuno-GO-Au/QD-QD nanotags, and (c) principle of GO-Au/QD-
QD-based ICA for the simultaneous and ultrasensitive detection of H1N1 and S. pneumoniae.
X. Cheng et al.
Journal of Hazardous Materials 459 (2023) 132192
5
second layer of QDs, which formed a typical multilayer nanolm. The
magnied TEM images in Fig. 1e–g show the detailed surface
morphology of GO-Au, GO-Au/QD, and GO-Au/QD-QD, respectively,
which clearly demonstrates that the GO surface was covered by a ho-
mogeneous AuNPs interlayer with individual AuNPs diameter of
approximately 16 nm and numerous 12 nm CdSe@ZnS QDs coated onto
the AuNPs surface. The enlarged TEM image in Fig. 1h–i reveals that the
lattice spacings from the adsorbed Au and QD nanoparticle were 0.235
and 0.315 nm, respectively, which can be attributed to the (111) planes
of cubic-structured Au and CdSe. The SEM images in Fig. 1j–l clearly
display the rough surface morphologies of GO-Au, GO-Au/QD, and
GO-Au/QD-QD. The energy-dispersive X-ray spectroscopy (EDS) spec-
trum (Fig. S3) indicates the presence of obvious C, O, S, Zn, Se, Cd, Au
signals in a single GO-Au/QD-QD nanostructure with the corresponding
elemental composition of 90.27 %, 2.33 %, 2.04 %, 0.82 %, 0.28 %, 0.97
% and 3.29 %, respectively. High-angle annular dark-eld scanning
(HAADF) TEM image (Fig. 1m) and EDS mapping results (Fig. 1n) also
reveal the element distribution of GO-Au/QD-QD nanosheets. They
clearly show that the Cd (blue), Se (purple), Zn (orange), and S (yellow)
elements were distributed on the outer surface of GO-Au nanosheet (C
Fig. 1. Characterization of the synthesized GO-Au/QD-QD nanoakes. TEM images of (a) GO, (b) GO-Au, (c) GO-Au/QD, and (d) GO-Au/QD-QD nanoakes.
Enlarged TEM images of partial (e) GO-Au, (f) GO-Au/QD, and (g) GO-Au/QD-QD nanoakes. HRTEM images of the adsorbed AuNPs and CdSe@ZnS QDs on (h) GO-
Au/QD, and (i) GO-Au/QD-QD nanoake. SEM images of (j) GO-Au, (k) GO-Au/QD, and (l) GO-Au/QD-QD nanoake. (m) HAADF-TEM image and (n) EDS elemental
(C, Au, Cd, Se, Zn, S) mapping image of a GO-Au/QD-QD nanoake.
X. Cheng et al.
Journal of Hazardous Materials 459 (2023) 132192
6
and Au elements). The zeta potentials of the nanosheets from each stage
are shown in Fig. S1, and the regular changes in surface potential
indicate that the fabrication of GO-Au/QD-QD was driven by electro-
static interaction. The DLS results display that the diameters of GO,
GO-Au, and GO-Au/QD-QD were in the range of 300–500 nm (Fig. 2d).
This nding suggests that the grafting of small AuNPs and QDs onto GO
surface does not increase the size of nanosheet obviously. These results
conrm that the designed lm-like dual-signal nanotag has been suc-
cessfully synthesized.
The colorimetric and uorescence properties of GO-Au/QD-QD
nanosheets were next evaluated. As exhibited in Fig. 2a, GO and GO-
PEI were both pale yellow under the sunlight, which conrms that the
PEI coating had no inuence on the color of GO. By coating AuNPs of
16 nm as color component, the GO-Au nanosheet exhibited purplish-red
color compared with GO solution. After the adsorption of the rst layer
and the second layer of QDs, the color of GO-Au/QD and GO-Au/QD-QD
remained purplish-red, which suggests that their colorimetric ability
was unaffected by QDs. UV–vis spectra results (Fig. 2b) show that the
AuNPs-loaded GO nanosheets (GO-Au, GO-Au/QD, and GO-Au/QD-QD)
all possessed a strong absorption peak around 538 nm, while the GO and
GO-PEI had no absorption peak in the same region. Obviously, the UV
absorption of dual-signal nanosheet was caused by the plasmonic reso-
nance excitation of the inner layer of AuNPs. The uorescence photo-
graphs and uorescence spectra in Fig. 2a and c indicate that only the
QDs-coated nanosheets can produce distinct red uorescence and the
GO-Au/QD-QD containing more QDs emitted much stronger lumines-
cence than GO-Au/QD. This result conrms that the strong colorimetric
signal and superior uorescence signal of proposed nanosheet can be
easily achieved by successive adsorption of AuNPs and two layers of QDs
onto its surface.
Notably, the rst interlayer of GO-Au/QD-QD nanoakes was formed
by mixing AuNPs and QDs. Thus, the ratio of two subassemblies should
be optimized to obtain the best performance. The detailed optimization
procedure is shown in Supporting information S1 and Fig. S4–S5. The
colloidal and optical stabilities of GO-Au/QD-QD nanosheets in harsh
environment were carefully investigated. As displayed in Fig. 2e–f, the
dual-signal GO-Au/QD-QD nanosheets showed excellent colloidal sta-
bility and colorimetric signal stability in aqueous solutions with a wide
pH range of 1–14 and high salt samples (0–1000 mM NaCl). Moreover,
the uorescence signal of GO-Au/QD-QD remained stable in the com-
plex samples except strong acidic solution (pH <2). And there are also
no obvious changes in the colloidal stability and uorescence intensity
of GO-Au/QD-QD after 90 days of storage under dark condition
(Fig. S6). These results suggest that the proposed GO-Au/QD-QD
nanosheet perfectly combined the stability and dispersity of GO nano-
akes, the excellent colorimetric performance of AuNPs, and the supe-
rior uorescence performance of QDs. These properties gave them
tremendous power to replace common colloidal gold and QDs as a high-
performance ICA nanoprobe.
3.3. Construction of GO-Au/QD-QD-based ICA biosensor
The construction of the GO-Au/QD-QD-based dual-signal ICA
biosensor for H1N1 and S. pneumoniae is shown in Scheme 1c, which
consists of three parts including a sample pad, an absorbent pad, and a
NC membrane with two test lines. The H1N1 and S. pneumoniae anti-
bodies were correspondingly dispensed onto the NC membrane to
construct one virus test line and one bacteria test line. Next, the cross-
reaction experiment of GO-Au/QD-QD-based ICA biosensor was rst
conducted by testing samples containing a mixture of H1N1/
S. pneumoniae, 10
4
cells/mL of S. pneumoniae, 10
6
copies/mL of H1N1,
and none of H1N1/S. pneumoniae. As shown in the image of Fig. 3a, the
GO-Au/QD-QD-ICA strips exhibited two purple T lines under natural
light and two bright uorescence T lines under UV light when the
sample contained two target detections. By contrast, only one purple
and uorescence T line appeared when the sample only contained
Fig. 2. Properties of dual-signal GO-Au/QD-QD nanoakes. (a) photographs, (b) UV–vis spectra, and (c) uorescence emission spectra of GO, GO-PEI, GO-Au, GO-
Au/QD, and GO-Au/QD-QD nanoakes. (d) DLS distribution of GO, GO-Au, and GO-Au/QD-QD nanoakes. (e) Colorimetric and uorescence stability of GO-Au/QD-
QD nanoakes in aqueous solutions with different pH values. (f) Colorimetric and uorescence stability of GO-Au/QD-QD nanoakes in various salt concentrations
(0–1000 mM NaCl).
X. Cheng et al.
Journal of Hazardous Materials 459 (2023) 132192
7
S. pneumoniae or H1N1, and no false positive signals appeared in the
strips when containing no target pathogenic microorganism. The fluor-
escence intensity values of the two T lines of ICA strips were simulta-
neously measured by utilizing the commercial uorescence strip reader,
and the results are shown in Fig. 3b. These results indicate the lack of
cross-reaction between S. pneumoniae and H1N1 antibodies and provide
the foundation for simultaneous and quantitative determination of
H1N1 and S. pneumoniae. Fig. 3c–e displayed the interior morphology of
the test zones from a positive test and a negative control, respectively.
The SEM images in Fig. 3c–e taken from the corresponding test lines
clearly illustrate the presence of many lm-like GO-Au/QD-QD nano-
akes on the viral test line and many S. pneumoniae-GO-Au/QD-QD
complexes on the bacterial test line for the positive sample. By contrast,
the same zone for blank control had no GO-Au/QD-QD nanoakes
(Fig. 3e). These results further conrm the reliability of our proposed
biosensor.
Herein, the GO-Au/QD-QD nanosheets were used as liquid nanotags
to directly mix with the tested sample solution and then loaded onto the
sample pad of strip to start the ICA detection. GO-Au/QD-QD nanosheets
have abundant carboxy groups of QD shell on their surface. Thus, anti-
H1N1/S. pneumoniae antibodies can be efciently conjugated with GO-
Au/QD-QD nanoakes via EDC/NHS chemistry. Next, EDC/NHS
chemistry was introduced to conjugate anti-H1N1/S. pneumoniae anti-
body on the surface of GO-Au/QD-QD nanoakes and formed immuno-
GO-Au/QD-QD nanotags. To obtain the best detection performance,
some important factors of the biosensor, such as the anti-H1N1/
S. pneumoniae antibodies on the GO-Au/QD-QD nanotags, the concen-
trations of the H1N1/S. pneumoniae antibodies sprayed on T lines,
chromatographic reaction time, and the FBS ratio in running buffer were
well optimized. EDC/NHS chemistry was introduced to conjugate anti-
H1N1/S. pneumoniae antibody on the surface of GO-Au/QD-QD nano-
akes and formed immuno-GO-Au/QD-QD nanotags. The amount of
anti-H1N1/S. pneumoniae antibodies on the GO-Au/QD-QD nanoakes
was optimized. Fig. S7 shows that 12
μ
g anti-H1N1/S. pneumoniae an-
tibodies could reach saturation. The TEM images clearly reveal that the
immuno-GO-Au/QD-QD nanotags could quickly and effectively bind to
the S. pneumoniae through vortex for 10 s (Fig. S8a–b). The results
indicate the afnity of the immuno-GO-Au/QD-QD nanotags. We further
optimized the concentration of H1N1/S. pneumoniae antibodies modi-
ed on the T zones to achieve the highest sensitivity of the ICA system.
As shown in Fig. 3f–g, the combination of 1 mg/mL of H1N1 antibody
and 0.8 mg/mL of S. pneumoniae antibody achieved the highest SNR and
Fig. 3. (a) Photographs and (b) corresponding fluorescence intensity (T lines) of GO-Au/QD-QD-based ICA with different concentrations of H1N1 and S. pneumoniae:
(1) 10
6
copies/mL, 10
4
cells/mL; (2) 0 copies/mL, 10
4
cells/mL; (3) 10
6
copies/mL, 0 cells/mL; and (4) 0 copies/mL, 0 cells/mL. Typical SEM images of the cor-
responding test lines in the presence of (c) 10
6
copies/mL H1N1, (d) 10
4
cells/mL S. pneumonia and (e) blank control (PBS only). Optimization of (f) H1N1 and (g)
S. pneumoniae antibody concentration on the two T lines. (h) Optimization of FBS ratio in PBST for GO-Au/QD-QD-based ICA strips. The error bars indicate standard
deviations calculated from three separate tests.
X. Cheng et al.
Journal of Hazardous Materials 459 (2023) 132192
8
was chosen to modify on the T1 and T2 lines, respectively. Our previous
studies have shown that PBS is a reliable buffer, Tween-20 could make
immune complexes ow smoothly on the strip, and FBS is a key factor in
reducing nonspecic adsorption of QD nanotags [21,41]. Thus, the FBS
ratio in PBST was optimized. As demonstrated in Fig. 3h, using PBST-F
(10 mM PBS containing 1 % Tween and 4 % FBS) as the running buffer
effectively suppressed the nonspecic adsorption on the T lines and
achieved the maximum SNR. Finally, we further assessed the inuence
of chromatographic reaction time of our ICA by analyzing the SNR value
of T lines. Fig. S9 shows that 20 min was sufcient for our
GO-Au/QD-QD-based ICA.
3.4. Analytical performance of the GO-Au/QD-QD-based ICA
The performance of the GO-Au/QD-QD-based ICA simultaneous
detection of bacteria and virus (H1N1 and S. pneumoniae) was evaluated.
Precise concentrations of the prepared H1N1 virus and S. pneumoniae
samples were determined using ddPCR and the plate colony counting
method, respectively. The results of ddPCR and bacteria culture in plates
are displayed in Fig. S10–S11. A gradient dilution method was used to
prepare samples with different concentration gradients (H1N1: 0–10
7
copies/mL, S. pneumoniae: 0–10
5
cells/mL) and the sensitivity was
determined under optimized conditions. The colorimetric and uores-
cence images of the tested strips for simultaneous determination of
H1N1 and S. pneumoniae are exhibited in Fig. 4a–b. As observed, the
purple-red colorimetric signal and red uorescence signal on the two T
lines get deeper and brighter with the increasing concentrations of H1N1
and S. pneumoniae. The purple-red colorimetric signal on T lines could be
distinguished by the naked eye at the concentration of 5 ×10
4
copies/
mL (H1N1) and 5 ×10
2
cells/mL (S. pneumoniae). We also conducted
the traditional colloidal gold ICA method to detect H1N1 and
S. pneumoniae (Fig. 4i), in which the purple-red colorimetric signal LODs
were 5 ×10
5
copies/mL and 5 ×10
3
cells/mL, respectively. Thus, the
sensitivity of the GO-Au/QD-QD-ICA in the colorimetric mode for
simultaneous detection of H1N1 and S. pneumoniae is 10 times higher
than that of traditional colloidal gold ICA. The promotion in sensitivity
Fig. 4. Photographs of GO-Au/QD-QD-based ICA strips for H1N1 (T1) and S. pneumoniae (T2) simultaneous detection in running buffer under (a) natural light and
(b) UV light. (c) Detailed uorescence intensity on T areas of GO-Au/QD-QD-based ICA (upper) and GO-QD-based ICA (below) strips for simultaneous detection of
H1N1 and S. pneumoniae. Corresponding calibration curves of GO-Au/QD-QD-based ICA strips for (d) H1N1 and (e) S. pneumoniae. Photographs of (f) GO-QD-based
ICA and (i) AuNPs-based ICA strips for the same S. pneumoniae/H1N1 samples. Corresponding calibration curves of GO-QD-based ICA strips for (g) H1N1 and (h)
S. pneumoniae (i) Photographs of AuNPs-Bbased.
X. Cheng et al.
Journal of Hazardous Materials 459 (2023) 132192
9
may be conducted by the Au interlayer of the GO-Au/QD-QD nanosheet.
As displayed in Fig. 4b, the visible uorescence signal on the T lines
of GO-Au/QD-QD-ICA could be observed at the concentration of 10
4
copies/mL (H1N1) and 10
2
cells/mL (S. pneumoniae). We conducted the
GO-QD-ICA simultaneously detecting H1N1 and S. pneumoniae under
the same detection conditions for comparison (Fig. 4f). The visible
uorescence signal on the T lines of GO-QD-ICA could be observed at the
concentration of 5 ×10
4
copies/mL (H1N1) and 5 ×10
2
cells/mL
(S. pneumoniae), while GO-QD-ICA had no colorimetric signal. Thus, the
sensitivity of the GO-Au/QD-QD-ICA in visible uorescence signal is two
times higher than that of GO-QD-ICA. To conduct more accurate quan-
titative analysis of GO-Au/QD-QD-ICA, the uorescence intensities of
the T1 and T2 lines were read and plotted as a function of the logarithm
of H1N1 and S. pneumoniae concentrations (Fig. 4d–e). The uorescence
intensity of the two T lines for H1N1 and S. pneumoniae exhibited a wide
dynamic range, and the correlation coefcients (R
2
) were around 0.994
and 0.999. Based on the established tting curve, LODs of the GO-Au/
QD-QD-ICA for H1N1 and S. pneumoniae simultaneous detection
reached 891 copies/mL and 17 cells/mL, respectively, according to the
triple standard deviation of the blank control. Fig. 4 g–h shows the
corresponding calibration curves of GO-QD-based ICA strips for H1N1
and S. pneumoniae for comparison, and the LODs of GO-QD-ICA for
simultaneous detection of H1N1 and S. pneumoniae reached 10
4
copies/
mL and 10
2
cells/mL, respectively. Fig. 4c shows the uorescence in-
tensity comparison between the GO-Au/QD-QD-ICA (upper) and the
GO-QD-ICA (below) simultaneous detection of H1N1 and S. pneumoniae.
The above mentioned results show that the sensitivity of the GO-Au/QD-
QD-ICA for simultaneous detection of H1N1 and S. pneumoniae is 500
times higher than that of traditional colloidal gold ICA, and it is 10 times
higher than that of GO-QD-ICA. These results indicate that the dual-
signal mode biosensor could greatly broaden the applied range of the
ICA method. Its colorimetric mode allows for quick determination of
virus/bacteria, and its uorescence mode ensures the highly sensitive
and quantitative detection of target pathogens.
3.5. Analysis of specicity, repeatability, and accuracy of dual-mode GO-
Au/QD-QD-based ICA
The specicity and repeatability of the proposed method should also
be assessed. Four highly contagious respiratory viruses (u B, RSV,
SARS-CoV-2, and HAdV) and two common respiratory bacteria (Staph-
ylococcus aureus and Haemophilus inuenzae) were spiked into a running
buffer as interference pathogens to test the specicity of the established
method. The results displayed in Fig. S12 show that all interference
pathogens exhibited no colorimetric or uorescence signal on the T1 and
T2 zones. By contrast, the positive groups containing target pathogens
produced obvious colorimetric and uorescence signals on their corre-
sponding T zones. Then, the stability of the GO-Au/QD-QD-based ICA
was further evaluated. As shown in Fig. S13, six separate strips all
exhibited good colorimetric and uorescence signals reproducibility on
the T zones. Moreover, the relative standard deviation (RSD) values
were no more than 5.3 %. These results indicate the excellent specicity
and repeatability of the GO-Au/QD-QD-based ICA.
We further evaluated the accuracy of our proposed dual-mode bio-
sensors in real clinical and environmental samples of sputum, lake
water, and object surface. The object surface samples were collected
using medical sterile cotton swabs, which were randomly wiped on the
hospital door handles, tables, and chairs and immediately inserted into
sterile tubes with 1 ×running buffer (1000
μ
L). Different concentrations
of target pathogens (H1N1 with concentrations of 10
7
, 10
6
, 10
5
, and 10
4
copies/mL; S. pneumoniae with concentrations of 10
5
, 10
4
, 10
3
, and 10
2
cells/mL) were spiked into the complex specimens and conducted with
the GO-Au/QD-QD-ICA strips. Then, the proposed GO-Au/QD-QD-ICA
was used to test the sputum, lake water, and object surface samples
with xed concentrations of H1N1 and S. pneumoniae. The colorimetric/
uorescence images and corresponding uorescence intensity are
displayed in Fig. 5a–c. The changes in colorimetric and uorescence
signals were consistent with the changes in corresponding concentra-
tion. The detailed uorescence intensity of the strips was measured to
calculate the recoveries (Table 1). The average recoveries of GO-Au/QD-
QD-ICA for simultaneous detection of H1N1 and S. pneumoniae ranged
from 89.2 % to 104.0 % and from 91.2 % to 107.0 %, respectively. The
RSD values ranged from 3.0 % to 8.0 %. In addition, the freeze-dried
immuno-GO-Au/QD-QD tags and prepared test strips maintained sta-
ble detection performance for target bacteria/virus after storage for 90
days under room temperature, indicating the excellent long-term sta-
bility of our dual-mode ICA method (Fig. S14). Compared with other
recently reported highly sensitive ICA-based strategies for respiratory
virus or bacteria detection, the proposed GO-Au/QD-QD-ICA exhibited
better performance including broad application range and multifunc-
tional detection ability (Table S1). The performance of GO-Au/QD-QD-
ICA can be attributed to the advanced lm-like colorimetric/uores-
cence dual-signal nanotag. All the results conrm that the proposed
colorimetric/uorescence dual-mode GO-Au/QD-QD-ICA has good
applicability and accuracy in the on-site detection of respiratory path-
ogenic microorganisms including viruses and bacteria.
4. Conclusions
We developed a lm-like GO-Au/QD-QD-based dual-mode ICA
biosensor for ultrasensitive multiplexed screening of H1N1 and
S. pneumoniae in real environment and biological samples. The innova-
tive GO-Au/QD-QD nanolm consisted of four functional domains: (i) a
GO nanosheet of 300–500 nm as exible supporter to provide excellent
stability and dispersibility in samples solution, (ii) one layer of density-
controlled AuNPs to provide strong colorimetric signal, (ii) two layers of
QDs in the interlayer and outer shell to generate superior uorescence
signal, and (iv) surface-conjugated anti-H1N1/S. pneumoniae antibodies
that can efciently bind to the target virus/bacteria. Based on the high-
performance GO-Au/QD-QD tags, the developed dual-mode ICA method
allowed the rapid screening (colorimetry) and quantitative detection
(uorometry) of H1N1/S. pneumoniae within 20 min. Under colori-
metric mode, the visual detection limits of the proposed assay for H1N1
and S. pneumoniae reached 5 ×10
4
copies/mL and 5 ×10
2
cells/mL,
respectively. Under uorescence mode, the LODs of the proposed assay
for target virus and bacteria were down to 891 copies/mL and 17 cells/
mL, respectively, which are about 500 and 10 times higher than those of
AuNPs-based ICA and GO-QD-based ICA strips, respectively. The dual-
mode ICA also exhibited excellent performance in the detection of real
environment samples (lake water and object surface) and clinical sam-
ples (sputum). Therefore, it has great practical potential for on-site
detection of respiratory pathogenic microorganism including viruses
and bacteria.
Environmental Implication
The respiratory pathogenic microorganisms such as virus inuenza A
(H1N1) and respiratory bacteria Streptococcus pneumoniae
(S. pneumoniae) are highly resistant to the environment and can spread
rapidly through close person-to-person contact or environmental media
(e.g., air, water, and object surface). The proposed analytical method
can simultaneously detect H1N1 and S. pneumoniae in complex envi-
ronment samples and biological samples. This method holds great po-
tential for on-site monitoring of pathogenic microorganisms.
CRediT authorship contribution statement
Xiaodan Cheng: Methodology, Writing-original draft preparation.
Xingsheng Yang: Methodology. Zhijie Tu: Methodology. Zhen Rong:
Supervision. Chongwen Wang: Supervision, Conceptualization,
Writing – review & editing. Shengqi Wang: Supervision,
Conceptualization.
X. Cheng et al.
Journal of Hazardous Materials 459 (2023) 132192
10
Declaration of Competing Interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgements
This study was supported by the National Natural Science Founda-
tion of China (Grant no. 81902159, 81830101, 32200076), and the
Natural Science Foundation of Anhui Province (Grant no.
2208085MB29). The authors would like to thank Prof. Chengfeng Qin
from Beijing Institute of Microbiology and Epidemiology for providing
experimental materials, and thank Ms Le Zhao of National Center for
Nanoscience and Technology for helping to conduct SEM analysis.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.jhazmat.2023.132192.
References
[1] O’Horo J.C., Cawcutt K. Lower Respiratory Tract Infections - ScienceDirect. 2022.
[2] Li, Z.J., Zhang, H.Y., Ren, L.L., Lu, Q.B., Ren, X., Zhang, C.H., et al., 2021.
Etiological and epidemiological features of acute respiratory infections in China.
Nat Commun 12 (1), 5026. https://doi.org/10.1038/s41467-021-25120-6.
[3] Yao, L., Zhu, W., Shi, J., Xu, T., Qu, G., Zhou, W., et al., 2021. Detection of
coronavirus in environmental surveillance and risk monitoring for pandemic
control. Chem Soc Rev 50 (6), 3656–3676. https://doi.org/10.1039/d0cs00595a.
[4] Man, W.H., van Houten, M.A., M´
erelle, M.E., Vlieger, A.M., Chu, M.L.J.N.,
Jansen, N.J.G., et al., 2019. Bacterial and viral respiratory tract microbiota and
host characteristics in children with lower respiratory tract infections: a matched
case-control study. Lancet Resp Med 7 (5), 417–426. https://doi.org/10.1016/
s2213-2600(18)30449-1.
[5] Suleman, S., Shukla, S.K., Malhotra, N., Bukkitgar, S.D., Shetti, N.P., Pilloton, R.,
et al., 2021. Point of care detection of COVID-19: Advancement in biosensing and
diagnostic methods. Chem Eng J 414, 128759. https://doi.org/10.1016/j.
cej.2021.128759.
[6] Hariri, L.P., North, C.M., Shih, A.R., Israel, R.A., Maley, J.H., Villalba, J.A., et al.,
2021. Lung histopathology in coronavirus disease 2019 as compared with severe
acute respiratory sydrome and H1N1 inuenza: a systematic review. Chest 159 (1),
73–84. https://doi.org/10.1016/j.chest.2020.09.259.
[7] Suaya, J.A., Fletcher, M.A., Georgalis, L., Arguedas, A.G., McLaughlin, J.M.,
Ferreira, G., et al., 2021. Identication of Streptococcus pneumoniae in hospital-
acquired pneumonia in adults. J Hosp Infect 108, 146–157. https://doi.org/
10.1016/j.jhin.2020.09.036.
[8] Li, J., Lin, R., Yang, Y., Zhao, R., Song, S., Zhou, Y., et al., 2021. Multichannel
immunosensor platform for the rapid detection of SARS-CoV-2 and inuenza A
(H1N1) virus. ACS Appl Mater Interfaces 13 (19), 22262–22270. https://doi.org/
10.1021/acsami.1c05770.
[9] Tang, L., Gu, S., Gong, Y., Li, B., Lu, H., Li, Q., et al., 2020. Clinical signicance of
the correlation between changes in the major intestinal bacteria species and
COVID-19 severity. Eng-PRC 6 (10), 1178–1184. https://doi.org/10.1016/j.
eng.2020.05.013.
Fig. 5. Detection performance of GO-Au/QD-QD-based ICA strips for simultaneous detection of H1N1 and S. pneumoniae in real environment and clinical samples: (a)
sputum, (b) lake water, (c) object surface.
Table 1
Recovery efciency of H1N1 virions and S. pneumoniae in real clinical and environmental samples of sputum, lake water, and object surface using GO-Au/QD-QD-ICA.
Samples H1NI S. pneumoniae
Spiked Detected Recovery (%) RSD (%) Spiked Detected Recovery (%) RSD (%)
Sputum 1 ×10
7
8.92 ×10
6
89.2 4.0 1 ×10
5
9.17 ×10
4
91.7 7.6
1×10
6
1.04 ×10
6
104.0 5.5 1 ×10
4
1.02 ×10
4
102.0 3.0
1×10
5
8.97 ×10
5
89.7 7.3 1 ×10
3
1019 101.9 7.5
1×10
4
1.02 ×10
4
102.0 5.1 1 ×10
2
95 95.0 3.3
Lake water 1 ×10
7
9.26 ×10
6
92.6 5.1 1 ×10
5
9.94 ×10
4
99.4 8.0
1×10
6
9.83 ×10
5
98.3 6.0 1 ×10
4
1.07 ×10
4
107.0 4.1
1×10
5
9.66 ×10
4
96.6 4.9 1 ×10
3
950 95.0 4.5
1×10
4
9.61 ×10
3
96.1 6.1 1 ×10
2
102 102.0 6.0
Object surface 1 ×10
7
1.01 ×10
7
101.0 6.3 1 ×10
5
9.54 ×10
4
95.4 5.1
1×10
6
9.55 ×10
5
95.5 4.3 1 ×10
4
1.02 ×10
4
102.0 5.8
1×10
5
9.41 ×10
4
94.1 6.1 1 ×10
3
912 91.2 6.7
1×10
4
9.45 ×10
3
94.5 7.3 1 ×10
2
107 107 7.8
X. Cheng et al.
Journal of Hazardous Materials 459 (2023) 132192
11
[10] Xing, W., Liu, Y., Wang, H., Li, S., Lin, Y., Chen, L., et al., 2020. A high-throughput,
multi-index isothermal amplication platform for rapid detection of 19 types of
common respiratory viruses including SARS-CoV-2. Eng-PRC 6 (10), 1130–1140.
https://doi.org/10.1016/j.eng.2020.07.015.
[11] Ma, Y.-D., Chen, Y.-S., Lee, G.-B., 2019. An integrated self-driven microuidic
device for rapid detection of the inuenza A (H1N1) virus by reverse transcription
loop-mediated isothermal amplication. Sens Actuators B-Chem 296. https://doi.
org/10.1016/j.snb.2019.126647.
[12] Shen, J., Zhou, X., Shan, Y., Yue, H., Huang, R., Hu, J., et al., 2020. Sensitive
detection of a bacterial pathogen using allosteric probe-initiated catalysis and
CRISPR-Cas13a amplication reaction. Nat Commun 11 (1), 267. https://doi.org/
10.1038/s41467-019-14135-9.
[13] Charalampous, T., Kay, G.L., Richardson, H., Aydin, A., Baldan, R., Jeanes, C.,
et al., 2019. Nanopore metagenomics enables rapid clinical diagnosis of bacterial
lower respiratory infection. Nat Biotechnol 37 (7), 783–792. https://doi.org/
10.1038/s41587-019-0156-5.
[14] Cui, F., Zhou, H.S., 2020. Diagnostic methods and potential portable biosensors for
coronavirus disease 2019. Biosens Bioelectron 165, 112349. https://doi.org/
10.1016/j.bios.2020.112349.
[15] Jin, J., Lin, J., Huang, Y., Zhang, L., Jiang, Y., Tian, D., et al., 2022. High sensitivity
ratiometric uorescence temperature sensing using the microencapsulation of
CsPbBr3 and K2SiF6:Mn4+phosphor. Chin Chem Lett 33 (11), 4798–4802.
https://doi.org/10.1016/j.cclet.2022.01.017.
[16] Liu, L., Zhu, S., Sun, J., Xia, M., Zhao, Xe, Xu, G., 2021. Ratiometric uorescence
detection of bleomycin based on proximity-dependent uorescence conversion of
DNA-templated silver nanoclusters. Chin Chem Lett 32 (2), 906–909. https://doi.
org/10.1016/j.cclet.2020.07.015.
[17] Pang, Y., Li, Q., Wang, C., zhen, S., Sun, Z., Xiao, R., 2022. CRISPR-cas12a
mediated SERS lateral ow assay for amplication-free detection of double-
stranded DNA and single-base mutation. Chem Eng J 429. https://doi.org/
10.1016/j.cej.2021.132109.
[18] Liu, Y., Zhan, L., Qin, Z., Sackrison, J., Bischof, J.C., 2021. Ultrasensitive and
highly specic lateral ow assays for point-of-care diagnosis. ACS Nano 15 (3),
3593–3611. https://doi.org/10.1021/acsnano.0c10035.
[19] Wang, W., Yu, Q., Zheng, S., Li, J., Wu, T., Wang, S., et al., 2023. Ultrasensitive and
simultaneous monitoring of multiple small-molecule pollutants on an
immunochromatographic strip with multilayered lm-like uorescent tags. Sci
Total Environ 878, 162968. https://doi.org/10.1016/j.scitotenv.2023.162968.
[20] Parolo, C., Sena-Torralba, A., Bergua, J.F., Calucho, E., Fuentes-Chust, C., Hu, L.,
et al., 2020. Tutorial: design and fabrication of nanoparticle-based lateral-ow
immunoassays. Nat Protoc 15 (12), 3788–3816. https://doi.org/10.1038/s41596-
020-0357-x.
[21] Yang, X., Yu, Q., Cheng, X., Wei, H., Zhang, X., Rong, Z., et al., 2023. Introduction
of multilayered dual-signal nanotags into a colorimetric-uorescent coenhanced
immunochromatographic assay for ultrasensitive and exible monitoring of SARS-
CoV-2. ACS Appl Mater Interfaces 15 (9), 12327–12338. https://doi.org/10.1021/
acsami.2c21042.
[22] Huang, Z., Peng, J., Han, J., Zhang, G., Huang, Y., Duan, M., et al., 2019. A novel
method based on uorescent magnetic nanobeads for rapid detection of
Escherichia coli O157:H7. Food Chem 276, 333–341. https://doi.org/10.1016/j.
foodchem.2018.09.164.
[23] Loynachan, C.N., Thomas, M.R., Gray, E.R., Richards, D.A., Kim, J., Miller, B.S.,
et al., 2018. Platinum nanocatalyst amplication: redening the gold standard for
lateral ow immunoassays with ultrabroad dynamic range. ACS Nano 12 (1),
279–288. https://doi.org/10.1021/acsnano.7b06229.
[24] Tu, J., Wu, T., Yu, Q., Li, J., Zheng, S., Qi, K., et al., 2023. Introduction of
multilayered magnetic core–dual shell SERS tags into lateral ow immunoassay: a
highly stable and sensitive method for the simultaneous detection of multiple
veterinary drugs in complex samples. J Hazard Mater 448, 130912. https://doi.
org/10.1016/j.jhazmat.2023.130912.
[25] Wang, C., Wang, C., Li, J., Tu, Z., Gu, B., Wang, S., 2022. Ultrasensitive and
multiplex detection of four pathogenic bacteria on a bi-channel lateral ow
immunoassay strip with three-dimensional membrane-like SERS nanostickers.
Biosens Bioelectron 214, 114525. https://doi.org/10.1016/j.bios.2022.114525.
[26] Wang, J., Jiang, C., Jin, J., Huang, L., Yu, W., Su, B., et al., 2021. Ratiometric
uorescent lateral ow immunoassay for point-of-care testing of acute myocardial
infarction. Angew Chem 60 (23), 13042–13049. https://doi.org/10.1002/
anie.202103458.
[27] Han, H., Wang, C., Yang, X., Zheng, S., Cheng, X., Liu, Z., et al., 2022. Rapid eld
determination of SARS-CoV-2 by a colorimetric and uorescent dual-functional
lateral ow immunoassay biosensor. Sens Actuat B-Chem 351, 130897. https://
doi.org/10.1016/j.snb.2021.130897.
[28] Hu, J., Jiang, Y.Z., Wu, L.L., Wu, Z., Bi, Y., Wong, G., et al., 2017. Dual-signal
readout nanospheres for rapid point-of-care detection of Ebola virus glycoprotein.
Anal Chem 89 (24), 13105–13111. https://doi.org/10.1021/acs.
analchem.7b02222.
[29] Liu, X., Wang, K., Cao, B., Shen, L., Ke, X., Cui, D., et al., 2021. Multifunctional
Nano-sunowers With Color-magnetic-Raman properties for multimodal lateral
ow immunoassay. Anal Chem 93 (7), 3626–3634. https://doi.org/10.1021/acs.
analchem.0c05354.
[30] Ren, S., Li, Q., Wang, J., Fan, B., Bai, J., Peng, Y., et al., 2021. Development of a
fast and ultrasensitive black phosphorus-based colorimetric/photothermal dual-
readout immunochromatography for determination of noroxacin in tap water and
river water. J Hazard Mater 402, 123781. https://doi.org/10.1016/j.
jhazmat.2020.123781.
[31] Huang, L., Jin, J., Ao, L., Jiang, C., Zhang, Y., Wen, H.M., et al., 2020. Hierarchical
plasmonic-uorescent labels for highly sensitive lateral ow immunoassay with
exible dual-modal switching. ACS Appl Mater Interfaces 12 (52), 58149–58160.
https://doi.org/10.1021/acsami.0c18667.
[32] Wang, C., Yang, X., Gu, B., Liu, H., Zhou, Z., Shi, L., et al., 2020. Sensitive and
simultaneous detection of SARS-CoV-2-specic IgM/IgG using lateral ow
immunoassay based on dual-mode quantum dot nanobeads. Anal Chem 92 (23),
15542–15549. https://doi.org/10.1021/acs.analchem.0c03484.
[33] Chen, M., Liu, D., Du, X., Lo, K.H., Wang, S., Zhou, B., et al., 2020. 2D materials:
excellent substrates for surface-enhanced Raman scattering (SERS) in chemical
sensing and biosensing. TrAC-Trend Anal Chem 130, 115983. https://doi.org/
10.1016/j.trac.2020.115983.
[34] Morales-Narvaez, E., Baptista-Pires, L., Zamora-Galvez, A., Merkoci, A., 2017.
Graphene-based biosensors: going simple. Adv Mater 29 (7). https://doi.org/
10.1002/adma.201604905.
[35] Shen, W., Wang, C., Zheng, S., Jiang, B., Li, J., Pang, Y., et al., 2022. Ultrasensitive
multichannel immunochromatographic assay for rapid detection of foodborne
bacteria based on two-dimensional lm-like SERS labels. J Hazard Mater 437,
129347. https://doi.org/10.1016/j.jhazmat.2022.129347.
[36] Cheng, X., Zheng, S., Wang, W., Han, H., Yang, X., Shen, W., et al., 2021. Synthesis
of two-dimensional graphene oxide-uorescent nanoprobe for ultrasensitive and
multiplex immunochromatographic detection of respiratory bacteria. Chem Eng J
426, 131836. https://doi.org/10.1016/j.cej.2021.131836.
[37] Wang, W., Yang, X., Rong, Z., Tu, Z., Zhang, X., Gu, B., et al., 2023. Introduction of
graphene oxide-supported multilayer-quantum dots nanolm into multiplex lateral
ow immunoassay: A rapid and ultrasensitive point-of-care testing technique for
multiple respiratory viruses. Nano Res 16 (2), 3063–3073. https://doi.org/
10.1007/s12274-022-5043-6.
[38] Aziz, M.A., Kim, J.-P., Oyama, M., 2014. Preparation of monodispersed
carboxylate-functionalized gold nanoparticles using pamoic acid as a reducing and
capping reagent. Gold Bull 47 (1–2), 127–132. https://doi.org/10.1007/s13404-
014-0134-0.
[39] Ramírez-Ayala, M.F., Herrera-Gonz´
alez, A.M., Trejo-Carbajal, N., Guerrero, A.L.,
Vargas-Ramírez, M., García-Serrano, J., 2022. One-step synthesis and stabilization
of Au, Ag and Au-Ag nanoparticles with an ion-exchange polymer contained amide
and carboxylic acid functional groups. Colloid Surf A 647. https://doi.org/
10.1016/j.colsurfa.2022.129069.
[40] Fang Chen, X.L., Hihath, Joshua, Huang, Zhifeng, Tao, Nongjian, 2006. Effect of
anchoring groups on single-molecule conductance: comparative study of thiol-,
amine-, and carboxylic-acid-terminated molecules. JACS 128 (49), 15874–15881.
https://doi.org/10.1021/ja065864k.
[41] Wang, C., Cheng, X., Liu, L., Zhang, X., Yang, X., Zheng, S., et al., 2021.
Ultrasensitive and simultaneous detection of two specic SARS-CoV-2 antigens in
human specimens using direct/enrichment dual-mode uorescence lateral ow
immunoassay. ACS Appl Mater Interfaces 13 (34), 40342–40353. https://doi.org/
10.1021/acsami.1c11461.
X. Cheng et al.
