FRET-Created Traffic Light Immunoassay Based on Polymer Dots for PSA Detection

Article abstract of DOI:10.1021/acs.analchem.9b04747

There have been enormous efforts for developing the next generations of fluorometric lateral flow immunochromatographic strip (ICTS) owing to the great advances in fluorescent materials in these years. Here we developed one type of fluorometric ICTS based on ultrabright semiconducting polymer dots (Pdots) in which the traffic light-like signals were created by energy transfer depending on the target concentration. This platform was successfully applied for qualitatively rapid screening and quantitatively precise analysis of prostate-specific antigen (PSA) in 10 min from merely one drop of the whole blood sample. This FRET-created traffic light ICTS possesses excellent specificity and an outstanding detection sensitivity of 0.32 ng/mL for PSA. Moreover, we conducted proof-of-concept experiments to demonstrate its potential for multiplexed detection of cancer biomarkers at the same time in an individual test strip by taking advantage of the traffic light signals. To the best of our knowledge, it is the first model of a traffic light-like immunoassay test strip based on Pdots with multiplexing ability. These results would pave an avenue for designing the next generation of point-of-care diagnostics.

Full text of DOI:10.1021/acs.analchem.9b04747

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Article
FRET-Created Traffic Light Immunoassay
Based on Polymer Dots for PSA Detection
Yong-Quan Yang, Yu-Chi Yang, Ming-Ho Liu, and Yang-Hsiang Chan
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b04747 • Publication Date (Web): 09 Dec 2019
Downloaded from pubs.acs.org on December 10, 2019
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FRET-Created Traffic Light Immunoassay Based on Polymer
Dots for PSA Detection
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a
a
a
abc
Yong-Quan Yang, Yu-Chi Yang, Ming-Ho Liu, and Yang-Hsiang Chan*
aDepartment of Applied Chemistry, National Chiao Tung University, Hsinchu, Taiwan 30010
bCenter for Emergent Functional Matter Science, National Chiao Tung University, Hsinchu 30010, Taiwan
cDepartment of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan 80708
ABSTRACT: There have been enormous efforts for developing next generations of fluorometric lateral flow
immunochromatographic strip (ICTS) owing to the great advances in fluorescent materials in these years. Here we developed one
type of fluorometric ICTS based on ultrabright semiconducting polymer dots (Pdots) in which the traffic light-like signals were
created by energy transfer depending on the target concentration. This platform was successfully applied for qualitatively rapid
screening and quantitatively precise analysis of prostate-specific antigen (PSA) in 10 min from merely one drop of the whole blood
sample. This FRET-created traffic light ICTS possesses the excellent specificity and an outstanding detection sensitivity of 0.32
ng/mL for PSA. Moreover, we conducted proof-of-concept experiments to demonstrate its potential for multiplexed detection of
cancer biomarkers at the same time in an individual test strip by taking advantage of the traffic light signals. To the best of our
knowledge, it is the first model of traffic light-like immunoassay test strip based on Pdots with multiplexing ability. These results
would pave a avenue for designing the next generation of point-of-care diagnostics.
INTRODUCTION
Point-of-care (POC) detection, one type of in vitro
diagnostic test, can provide reliable and accurate testing
reporter for colorimetric analysis in which the subtle variations
of target concentrations are very difficult to be differentiated
from the shades of Au nanoparticles. This limits their
detection sensitivity as well as the quantitative ability.
Fluorometric ICTS can cleverly circumvent the low sensitivity
1
-3
results in a timely manner. As compared to traditional
clinical diagnosis in a hospital which usually requires
sophisticated instruments and professional operators, POC
testing only needs to follow the simple instructions to obtain
the test results in seconds to hours. Besides, most POC kits
require merely small sample volumes (e.g., 1 L - 1 mL) of
complex physiological media to determine the target analytes
in the range of picomolar to millimolar concentrations. This
easy-to-handle, user-friendly, and cost-effective diagnostic
approach is very suitable for on-the-spot patient care or in-
home self-monitoring.
and quantitative concern of conventional colorimetric ICTS by
5, 13-19
employing fluorophores
(e.g., small organic dyes, Au
nanoclusters, up-converting nanoparticles, and quantum dots)
as reporters. Among these fluorescent probes, inorganic
quantum nanocrystals are the most extensively used one
2
0-22
because of their exceptional photophysical properties.
However, quantum dots suffer from several issues like
detrimental toxicity and active interactions with metal
23-26
ions/biothiols in body fluids.
Therefore, exploitation of an
ideal signal reporter remains an open challenge for
fluorometric ICTS.
Lateral-flow immunochromatographic assays or the
immunochromatography test strip (ICTS), is one of the most
prevalent type of POC, which was ever developed by
Unipath's PERSONA to measure luteinizing hormone and
estriol-3-glucuronide in urine. Nowadays, ICTS has been
27-28
In our previous works,
we have successfully utilized
TM
highly bright semiconducting polymer nanoparticles (Pdots) as
the fluorescent reporter in fluorometric ICTS for quantitative
sensing of tumor antigens with sensitivity of about 100 times
better than traditional colorimetric ICTS. The excellent
performance of Pdot-based ICTS is attributed to the
4
widely employed in diverse aspects, including clinical
diagnostics, environmental inspection, forensic investigation,
1-3,
food safety/drug residue inspection, and health supervision.
29-49
5
-12
advantageous properties of Pdots:
(i) exceptional
Despite of the impressive and flourishing development of
fluorescence brightness (over three orders of magnitude
brighter than small organic molecules and quantum
nanocrystals), offering high signal-to-noise ratios with
minimal background signal; (ii) high colloidal stability in body
ICTS, there are still several unmet challenges that might
hinder its widespread use. For example, most ICTS platforms
only allow for qualitative or semiquantitative analysis.
Additionally, the detection sensitivity of ICTS is only
adequate as compared to the most commonly used labeled
immunoassay techniques, enzyme-linked immunosorbent
assay (ELISA). Recently, enormous efforts have been exerted
on promoting detection sensitivity of ICTS by exploring new
types of signal reporting reagents. In contrast to traditional
50-53
fluids;
(iii) facile surface functionalization to conjugate
with corresponding antibodies for specific immune detection;
and (iv) amplified energy transfer in Pdots to enable the
creation of multicolor emissions under single excitation
wavelength, allowing for the design of multiplexed sensing
systems with visual recognition functions.
2
ICTS to utilize colloidal Au nanoparticles as the signal
1
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In this work, we intended to design the first example of
fluorescent coumarin derivatives, CA, was synthesized on the
basis of the previously reported literatures with revised
procedures.
fluorescence resonance energy transfer (FRET)-based ICTS by
exploiting the amplified energy in Pdots. Specifically, we
would like to produce a change of the emission color on the
test line depending on the concentration of the target analyte
54-56
Synthesis of ethyl 7-(diethylamino)-2-oxo-2H-chromene-
54
(
prostatic specific antigen (PSA), a cancer biomarker of
3-carboxylate, 1. In a round glass flask was added 1.0 g
(5.17 mmol) of 4-(diethylamino)-2-hydroxybenzaldehyde,
1.66 g (10.4 mmol) of diethyl malonate, and 0.2 mL (2.02
mmol) of piperidine in 6 mL of ethanol. The mixed solution
was heated to 80 °C and refluxed for 8 h. After the reaction,
the ethanol was removed by rotary evaporation and then the
prostate cancer, in this study). The shift of the emission color
on the test zone can be used for the prompt qualitative
recognition and the quantitative analysis of PSA in whole-
blood samples. As compared to traditional fluorometric ICTS
with only one individual emission color, FRET-based ICTS
with two or more emission colors embraces multiple unique
advantages: (i) multiple emission colors can be used create a
traffic light-like signal for rapid determination of the PSA
levels by naked eyes; (ii) amplified energy transfer in Pdots
results in the high FRET efficiency, allowing for the very
sensitive determination of PSA concentration; and (iii)
multiplexed detection capability benefited from the multicolor
emissions. The innovative model of FRET-based ICTS could
pave a new way in designing future generations of POC
diagnostics.
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residue was added with CH
times. The CH Cl layer was separated, dried by magnesium
sulfate, and then the CH Cl was evaporated by the rotary
2 2
Cl to extract with water for three
2
2
2
2
evaporator and vacuum system. The obtained yellow-brown
oily compound was then purified using column
chromatography on aluminum oxide, eluting by a mixture of
CH
2 2
Cl /ethyl acetate (10:1, v/v) solutions to get 997 mg (67 %)
1
of compound 1 as yellow oil. H NMR (400 MHz, CDCl
3
): δ =
8.43 (s, 1H), 7.35 (d, J = 7.8 Hz, 1H), 6.60 (d, J = 7.8 Hz, 1H),
6.46 (s, 1H), 4.37 (q, J = 8.2 Hz, 2H), 3.44 (q, J = 3.8 Hz, 4H),
1
.39 (t, J = 7.5 Hz, 3H), 1.23 (t, J = 7.3 Hz, 6H).
EXPERIMENTAL SECTION
Synthesis of 7-(diethylamino)-2-oxo-2H-chromene-3-
54
Chemicals. Deionized water (18.2 M•cm) was applied in
all of the experiments. Reaction reagents such as methanol,
ethanol, hexane, ethyl acetate, acetone, piperidine,
carboxylic acid, 2. In a round flask into which 0.65 g (0.26
mmol) of compound 1 in 6 mL 0.5 M NaOH dissolved in
methanol were added. The reaction was stirred at room
temperature for 16 h and then neutralized with 4 mL of 1 M
HCl. The mixture was kept at 4 °C for 24 h and the resulting
precipitates were collected. The crude product was then
purified via reprecipitation by using methanol/hexane to
dichloromethane
(CH
2
Cl
2
),
4,7,10-trioxa-1,13-
tridecanediamine, n-hydroxysuccinimide (NHS), polyethylene
glycol, octyl phenol ethoxylate, sodium hydroxide,
poly(styrene-co-maleic anhydride), cumene terminated
1
polymer with styrene of 75 wt. % (PSMA, M
tetrahydrofuran (THF), and N-(2-Hydroxyethyl)piperazine-N′-
2-ethanesulfonic acid) (HEPES) (CAS number 7365-45-9)
were purchased from Sigma-Aldrich. Methoxypolyethylene
n
~ 1900),
obtain 0.4 g (79 %) of compound 2 as an orange powder. H
3
NMR (400 MHz, CDCl ): δ = 12.3 (s, 1H), 8.66 (s, 1H), 7.45
(d, J = 9.7 Hz, 1H), 6.71 (d, J = 6.0 Hz, 1H), 6.53 (s, 1H), 3.47
(q, J = 4.0 Hz, 4H), 1.26 (t, J = 7.5 Hz, 6H).
(
glycol
tetrakis(triphenylphosphine)palladium(0),
albumin, saccharose, and
dimethylaminopropyl)carbodiimide (EDC) were obtained
from Alfa Asear. PF-TC6FQ (M ~ 19 500) polymer was
amine
(PEG1000,
M
w
=
1000),
tert-butyl (3-(3-aminopropoxy)propyl)carbamate, 3.55
bovine
serum
5
63 mg (2.56 mmol) of 4,7,10-trioxa-1,13-tridecanediamine
was dissolved in 20 mL of dry CH Cl . For the other flask,
305 mg (1.40 mmol) of di-tert-butyl dicarbonate was dissolved
in 20 mL of dry CH Cl . The solution containing di-tert-butyl
1-ethyl-3-(3-
2
2
n
37
prepared based on our previously published works.
2
2
Carboxylic acid-functionalized polystyrenes (PS-PEG-COOH,
dicarbonate was slowly added into the solution of 4,7,10-
trioxa-1,13-tridecanediamine in an ice water bath. The
reaction was further warmed to ambient temperature gradually
and kept stirring for 16 h. The mixture was then extracted with
3
product ID P18154-SEOCOOH, M
n
= 18.0x10 , M
w
/M
n
=1.09)
was obtained from Polymer Source, Inc. PSA proteins (30C-
CP1017U) and PSA primary antibodies (10-P20D and 10-
P20E) were purchased from Fitzgerald (MA, USA). Mouse
anti-human PSA primary antibodies were functionalized onto
the surfaces of coumarin nanoparticles (10-P20E, capture
antibody) and PF-TC6FQ Pdots (10-P20D, detection antibody),
respectively. AffiniPure goat anti-mouse IgG (H+L) secondary
antibodies (AB_2338447) were obtained from Jackson
ImmunoResearch Inc. (PA, USA) and modified onto the
control zone. These antibodies and antigens were diluted into
the desired concentrations with HEPES buffer (pH 7.4) and
then stably stored in the refrigerator for over 2 months.
Nitrocellulose membranes (pore size = 15 m, CNPC-SS12-
L2-P25), sample pads (GFB-R7L), conjugate substrates (PT-
R7), and absorbent matrices (AP 110) were got from
Advanced Microdevices Pvt. Ltd.. The NC membranes,
sample pads, conjugate pads, and absorbent pads were cut into
brine for three times and the CH
that, CH Cl was removed by the rotary evaporator and
vacuum system to yield 400 mg (49 %) of compound 3 as
2 2
Cl phase was isolated. After
2
2
1
colorless oil. H NMR (400 MHz, CDCl
3
): δ = 3.64-3.52 (m,
1
1
2H), 3.21 (t, J = 6.4Hz, 2H), 2.80 (t, J = 6.7 Hz, 2H), 1.80-
.69 (m, 4H), 1.43 (s, 9H).
Synthesis of tert-butyl (3-(3-(7-(diethylamino)-2-oxo-2H-
chromene-3-carboxamido)propoxy)propyl)carbamate, 4.56
In a round glass flask where 384 mg (1.2 mmol) of compound
3
, 261 mg (1.00 mmol) of compound 2, 288 mg (1.50 mmol)
of EDC, and 173 mg (1.50 mmol) of NHS in 2 mL of CH Cl
2
2
were injected. The mixed solution was stirred at ambient
temperature for 12 h. The organic phase was extracted with
water for 3 times and isolated, dried by anhydrous MgSO
CH Cl was further removed through rotary evaporation. The
obtained product was then refined using column
chromatography filled with Al , eluting with CH Cl /ethyl
4
.
2
2
6
0 x 4 mm, 1.4 x 4 mm, 6 x 4 mm, and 2.1 x 4 mm (length x
width), respectively. These above materials were finally
assembled together and put inside the plastic cassettes. Green
2
O
3
2
2
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acetate (1:1, v/v) to afford 295 mg (35 %) of compound 4 as
Antibody Conjugation of PF-TC6FQ Pdots and CA
Nanoparticles. For the bioconjugation of PF-TC6FQ Pdots, 2
mL of PF-TC6FQ, 40 L of 1 M HEPES, 40 L of 5% PEG
1
yellow oily liquid. H NMR (400 MHz, CDCl
3
): δ = 8.89 (s,
1
2
3
8
7
H), 8.69 (s, 1H), 7.42 (d, J = 8.9 Hz, 1H), 6.64 (dd, J = 9.1,
.6 Hz, 1H), 6.49 (s, 1H), 5.05 (s, 1H), 3.73-3.50 (m, 12H),
.45 (q, J = 7.0 Hz, 4H), 3.22 (t, J = 7.6 Hz, 4H), 1.90 (d, J =
.3 Hz, 2H), 1.75 (d, J = 8.3 Hz, 2H), 1.43 (s, 9H), 1.23 (t, J =
.1 Hz, 6H).
(M = 3015-3685), 40 L (1 mg/mL) of fresh-prepared EDC,
n
1
0 L (1 mg/mL) of NHS, and 60 L of PSA antibody (10-
P20D, 0.1 mg/mL) were added in a vial and further reacted for
h at ambient temperature. Then, 2 L (1 mg/mL) of
PEG1000 was added and stirred for another 20 min. After the
bioconjugation, the Pdot-PSA conjugates were refined by
Sephacryl (S-300 HR) size exclusion chromatography resins
to remove the free antibodies. The purified Pdot-PSA solution
was further concentrated by a Pall Omega centrifugal device
tube (molecular weight cut-off: 100 kDa) with the spinning
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Synthesis
(diethylamino)-2-oxo-2H-chromene-3-carboxamide, CA.
95 mg (0.52 mmol) of compound 4 was deprotected by
trifluoroacetic acid (20%) in CH Cl at room temperature for
2 h. The reaction was terminated by extracting with sodium
of
N-(3-(3-aminopropoxy)propyl)-7-
56
2
2
2
1
bicarbonate aqueous solution (10%) for 3 times. The organic
setting of 1500 rpm for 5 min to get 400 L of concentrated
Pdot-PSA solution.
For CA-PSA bioconjugates that intended to be fabricated on
solvent was then removed to obtain 217 mg (90 %) of
1
compound CA as brown liquid. H NMR (400 MHz, CDCl
3
):
δ = 9.05 (s, 1H), 8.68 (s, 1H), 8.26 (s, 2H), 7.55 (d, J = 9.0 Hz,
1H), 6.71-6.63 (m, 1H), 6.49 (s, 1H), 3.83-3.50 (m, 12H), 3.46
(q, J = 7.4 Hz, 4H), 3.35-3.32 (m, 4H), 2.05 (d, J = 8.4 Hz,
the test line, 200 L (1 mg/mL) of CA, 160 L of 1M HEPES,
1
60 L 5% PEG (M = 3015-3685), 6 L (1 mg/mL) of fresh-
n
prepared EDC, 1.5 L (1 mg/mL) of NHS, and 37.5 L of
PSA antibody (10-P20E, 0.1 mg/mL) were added in a 2 mL of
glass vial and let stir for 3 h. After the reaction, the CA-PSA
conjugates were purified and concentrated by a standard 17 x
00 mm conical-end centrifuge tube (Pall Macrosep®
Advance Centrifugal Devices, molecular weight cut-off: 10
kDa) to obtain a final volume of 25 L.
For CA-BSA bioconjugates that intended to be fabricated
on the control line, 8.0 mL (1 mg/mL) of CA, 160 L of 1M
2
H), 1.85 (d, J = 8.4 Hz, 2H), 1.24 (t, J = 7.0 Hz, 6H).
Preparation of Polymer Nanoparticles. The preparation
procedures of three Pdot stock THF solution were as follows:
PF-TC6FQ, compound CA, and PSMA separately dissolved in
THF with the concentration of 1000 ppm. 200 L of PF-
TC6FQ, 30 L of PS-PEG-COOH, and 10 L of PSMA were
taken out and mixed well in 5 mL THF solution. The main
purpose to use PS-PEG-COOH is to reduce the undesired
biomolecule adsorption because of its polyethylene glycol
units. PS-PEG-COOH can also provide the carboxylic units
but the percentage of the grafted COOH group is lower than
1
HEPES, 160 L 5% PEG (M = 3015-3685), 320 L (1
n
mg/mL) of fresh-prepared EDC, 80 L (1 mg/mL) of NHS,
and 160 L of bovine serum albumin (BSA) antibody (10
mg/mL) were added in a 20 mL of glass vial and let stir for 4 h.
After the reaction, the CA-BSA conjugates were purified and
concentrated by a standard 17 x 100 mm conical-end
centrifuge tube (Pall Macrosep® Advance Centrifugal Devices,
molecular weight cut-off: 10 kDa) to obtain a final volume of
400 L with 10% glycerin.
1
0 % based on the manufacturer’s analysis which might affect
the efficiency of surface bioconjugation. Therefore, we added
additional PSMA to offer the COOH terminal groups on the
Pdot surface for further antibody conjugation. The THF
mixture was then rapidly added to 10 mL of pure H
intense sonication for ~ 30 s. THF was further evaporated by
continuously blowing by N gas on a 80 °C for 30-40 min. The
2
O with
2
as-prepared Pdot aqueous solution was kept purging with
nitrogen gas at room temperature for 10 min and then passed
through a 220 nm polyether sulfone syringe filter. For each
batch, the final volume of the nanoparticle samples was tuned
to ca. 8 mL and checked with UV-vis spectroscopy to ensure
the fixed concentration of Pdots.
Fabrication of Immunochromatography Test Strips for
PSA Detection. The components of ICTS were modified in an
effort to minimize the nonspecific interference. For
nitrocellulose membranes, 0.05 % of glycerin aqueous
solution was used to soak the membranes for 1 h and then
dried under vacuum. For the pre-modification of conjugate
pads, 5 % of glycerin aqueous solution was used instead. To
fabricate the conjugate pads with Pdot probes, we first
prepared a solution containing 20 L of PF-TC6FQ Pdots, 15
Preparation of coumarin (CA) nanoparticles. Compound
CA was first conjugated with PSMA via EDC-catalyzed
reactions. Specifically, 46.4 mg (0.1 mmol) of CA, 38 mg

L of 20% sucrose, 5 L of 20 mM HEPES buffer, and 40 L
(
(
0.02 mmol) of PSMA, 38 mg (0.2 mmol) of EDC, and 23 mg
0.2 mmol) of NHS were added into 2 mL of dichloromethane
of 5% glycerin; and then dipped the conjugate pads into the
solution for 20 min. The modified conjugate pads were then
dried under vacuum for 10 min. For the preparation of test
samples, 0-30 L of PSA (0.1 ng/L), 32 L of 10% PEG, 48
and then stirred for 30 min. After that, 20 mg (0.02 mmol) of
PEG1000 was added and stirred for another 6 h. After the
reaction, the mixture was extracted with water for three times
and dichloromethane was removed by a rotary evaporator. The
crude product was then purified by reprecipitation with
dichloromethane/hexane to yield 50 mg of PSMA-grafted CA,
compound PCA as an orange-yellow solid. For the preparation
of CA nanoparticles, 200 L of CA (1 mg/mL in THF) and 30

L of 20 mM HEPES, and 10 % of human serum were mixed
together for measurements. Human serum was used to mimic
the physiological conditions of real samples and
simultaneously could greatly reduce the undesired
biomolecules binding. For the preparation of whole-blood
analytes, we utilized a one-time-use sterile blood lancet to
sting the fingertips to get one drop of fresh blood (5-10 L)
and then PSA proteins were added into the blood with the final
concentrations of 1-12 ng/mL. The blood was dropped inside
the sample port and then 80 L of a buffer solution containing

L of PS-PEG-COOH were first mixed in 5 mL of THF. The
CA-containing THF was further added to 10 mL of pure H
with intense sonication. THF was evaporated by continuously
purging dry N gas and then passed through a 220 nm
polyether sulfone syringe filter.
2
O
2
3
2 L of 10% PEG in 48 L of 20 mM HEPES was added
3
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inside the port promptly. While waiting for 10 min, the results
Selection and Optimization of Donor-Acceptor Pair. We
could be recorded by a Nikon D7500 digital camera under the
irradiation of 410 nm light irradiation with appropriate filters.
For the blue and red emission, a band-pass filter of 470 ± 25
nm and a long-pass filter of 600 nm were used, respectively.
The camera setting is: 1) F-stop: f/3.5; 2) Exposure time: 1/2-
wanted to create a FRET-based ICTS which can be excited by
an individual source with two distinct fluorescent colors. The
first aim was to select an appropriate donor-acceptor pair.
Originally we purported to select green fluorescent Pdots,
Poly[2,7-(9,9-dioctylfluorene)-alt-2,3-diphenylacrylonitrile]
(PFCN), as the donor and paired them with red fluorescent
Pdots, PF-TC6FQ, as the acceptor due to the substantial
spectral overlap between the emission spectrum of PFCN and
1
/8 s; 3) ISO speed: ISO-500; 4) Exposure bias: -1 step; 5)
Focal length: 18 mm; 6) Max aperture: 3.6; 7) Metering mode:
Multi-zone; 8) Flash mode: No flash; 9) 35 mm focal length:
27; 10) Color temperature: 4000 K.
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the absorption profile of PF-TC6FQ. However, we found
that PFCN Pdots could not be anchored onto the test line
probably due to their hydrophilic surface properties and lack
of anchoring groups on their surfaces. To tackle this issue, we
synthesized a blue-green fluorescent coumarin derivative (CA)
with an amine terminal group on the side chain as shown in
Scheme 1. The coumarin dye 1 was first synthesized with an
ethyl ester terminal moiety. The ethyl ester group was then
hydrolyzed by sodium hydroxide to afford the carboxyl
functional coumarin derivative 2. The compound 2 was
reacted with compound 2 through EDC-catalyzed coupling to
form compound 4. Finally, the tert-butoxycarbonyl group on
compound 4 was deprotected by trifluoroacetic acid to obtain
amine functional coumarin CA. We then conjugated
compound CA and methoxypolyethylene glycol amine
(PEG1000) with poly(styrene-co-maleic anhydride), cumene
terminated polymer with styrene (PSMA) via EDC-catalyzed
coupling. Here PSMA served as the polymer matrix to bind
with green-emitting CA, while PEG1000 was used to reduce
the non-specific adsorption. The ratio of CA to PEG1000 was
optimized in an effort to maximize the fluorescence brightness
and at the same time maintained the hydrophobic property of
the resulting polymers, PCA.
Characterization of FP/FG@Pdot Nanoparticles. The
average sizes of PF-TC6FQ Pdots and PCA nanoparticles
were measured using DLS instrument (Malvern Panalytical
Zetasizer Nano Series). Transmission electron microscope
images of PF-TC6FQ Pdots and PCA nanoparticles (diluted
over 100 times) were obtained by a bio-transmission electron
microscope (Hitachi, HT7700, accelerating voltage: 100-120
kV) The absorption spectra of Pdots and PCA nanoparticles
were acquired by using a USB4000 miniature spectrometer
(Ocean Optics, Inc.). Their corresponding emission spectra
were measured by a Hitachi F-4500 fluorometer with an
excitation wavelength at 400 nm. The fluorescence quantum
yields of PF-TC6FQ Pdots and PCA nanoparticles were
obtained using the integrating sphere detector in FS5-NIR
fluorometer to be 0.40 and 0.27, respectively.
RESULTS AND DISCUSSION
Our aim was to utilize two types of Pdots with two distinct
fluorescence colors (i.e., green and red fluorescence in this
work) and then functionalized the corresponding antibodies
onto the surface of Pdots. In the presence of target antigen,
these two kinds of Pdots would anchor together on the test line
of ICTS induced by the formation of the antibody-antigen
sandwich complex. In this scenario, FRET would occur from
the donor Pdots to the acceptor Pdots once the donor Pdots
were excited, causing the combinations of two emission colors
on the test line. The resulting emission color depends on the
concentration of the target antigen. Ideally, we would like to
create a traffic light-like signal based on FRET to mimic the
low-medium-high concentrations of the target antigen. For
example, the green emission represents the absence or low
concentration of the target because there exists no FRET
phenomenon. The intermediate color indicates the moderate
concentration of the target antigen, while the red color implies
the high concentration of the target antigen due to the obvious
FRET behavior. This new FRET-based ICTS allows the direct
qualitative visualization of the analyte from the emission color.
Quantitative measurement of the analyte concentration could
be further accomplished based on the relative intensities of
green to red fluorescence.
A
PF-TC6FQ
PCA
B
PF-TC6FQ
PCA
300
400
500
600
500
600
700
800
Wavelength(nm)
Wavelength(nm)
C 30 PCA nanoparticles
D 30 PF-TC6FQ Pdots
20
20
10
0
10
0
10
100
10
100
40
30
30
20
2
0
0
0
10
0
Scheme 1. Synthetic Routes of Coumarin Derivatives
1
10
100
10
100
Size (nm)
E
+
-
PCA
PCA-PSA/C
PF-TC6FQ PF-TC6FQ-PSA/D
Figure 1. (A) Absorption spectra of PCA nanoparticles (blue line)
and PF-TC6FQ Pdots (red line) in 20 mM HEPES buffer. The
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Scheme 2. Schematic Diagram Illustrating the Design of FRET-Created Traffic Light ICTSa
PCA
COOH
+
PS-PEG-COOH
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PS-PEG-COOH
COOH
Sample
PF-TC6FQ Pdots
PCA nanoparticles
PSA Antigen
PF-TC6FQ
FRET
Detection antibody
Capture antibody
IgG Antibody
Sample pad Conjugate pad
NC membrane Test Control
T) (C1) (C2)
Absorbent Pad
(
C2 C1
T
Safe
Caution
Danger
Negative
Positive
Positive
aFirst, coumarin derivative polymer (PCA) and semiconducting polymer (PF-TC6FQ) were synthesized and then mixed well in THF
with carboxylic acid terminal polystyrene (PS-PEG-COOH) separately. PCA nanoparticles and PF-TC6FQ Pdots were prepared via
nanoprecipitation by rapidly spiking the polymer THF mixtures into pure H O with intense sonication. PCA nanoparticles were further
2
conjugated with PSA capture antibodies while PF-TC6FQ Pdots were conjugated with PSA detection antibodies. The PSA-modified
PF-TC6FQ Pdots were fabricated onto the conjugate pad. There were two control lines on the nitrocellulose membrane: One was loaded
with PCA nanoparticles and the other was decorated with IgG secondary antibodies. The test line was modified with PSA-modified
PCA nanoparticles. This ICTS system allows the creation of traffic-light like signals based on FRET.
Surface Conjugation of PCA and PF-TC6FQ with
Antibodies. Once we identified the possible donor-acceptor
inset displays the photographic image of nanoparticle solutions in
daylight (left: PCA nanoparticles; right: PF-TC6FQ Pdots). (B)
pair, we tested the FRET-based test strip for PSA detection.
Fluorescence spectra of PCA nanoparticles (blue line) and PF-
As displayed in Scheme 2, the modified PSMA polymers,
TC6FQ Pdots (red line) in 20 mM HEPES buffer. The inset
PCA, were then co-precipitated with PS-PEG-COOH to
genereate COOH functionalized PCA-based polymer
nanoparticles. The PCA polymer nanoparticles were further
conjugated with capture antibodies for PSA antigen detection.
PF-TC6FQ Pdots were also prepared by nano-precipitation
and then conjugated with detection antibodies. The absorption
and fluorescence spectra of PCA nanoparticles and PF-TC6FQ
were displayed in Figure 1A and B. The broad emission peak
of PCA nanoparticles ranges from 490 nm to 600 nm, which
overlaps very well with the second absorption band of PF-
TC6FQ Pdots. This excellent spectral overlap between the
emission profile of the PCA and the absorption profile of the
PF-TC6FQ indicates that the FRET efficiency should be high
(vide infra). Based on the UV-visible spectra in Figure 1A, we
exhibits the photographic image of nanoparticle solutions excited
by a UV lamp (left: PCA; right: PF-TC6FQ). (C) Hydrodynamic
size distribution of PCA nanoparticles before (upper panel) and
after (bottom panel) PSA capture antibody conjugation. The inset
in the upper row represents the transmission electron microscopy
image of as-prepared PCA nanoparticles. The scale bars are 100
nm. (D) Hydrodynamic size distribution of PF-TC6FQ Pdots
before (upper panel) and after (bottom panel) PSA detection
antibody conjugation. The inset in the upper row represents the
transmission electron microscopy image of as-prepared PF-
TC6FQ Pdots. (E) Gel electrophoresis of bare PCA/PF-TC6FQ
nanoparticles and PSA-modified PCA/PF-TC6FQ nanoparticles.
PSA/C and PSA/D: Capture and detection antibodies.
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Control PSA CEA AFP
FBS RPMI HSA
1640
Control PSA CEA AFP FBS RPMI HSA
1640
Time (min)
Figure 2. Assessment on the targeting specificity of the FRET-based immunochromatographic assays. Photographic images of the test
strips after the detection of different analytes under 410 nm UV light (A) without filter, (B) with a band-pass filter of 470 ± 25 nm, and
(C) with a long-pass filter of 600 nm. The concentrations of the antigens (PSA, CEA, and AFP), FBS, and human serum albumin (HSA)
are 5 ng/mL, 5%, and 40 mg/mL, respectively. (D) Fluorescence ratios of the test zone to the second control zone at different times after
the liquid reached the absorbent pad. (E) Quantitative emission ratios of T/C1 in panel B as determined by ImageJ software. (F)
Quantitative emission ratios of T/C2 in panel C as calculated by ImageJ. The error bars in D-F show standard deviations obtained from
over 5 replicates.
selected an excitation source of 410 nm to directly excite the
PCA nanoparticles rather than the PF-TC6FQ Pdots. The
average hydrodynamic size of the as-prepared PCA
nanoparticles was determined by DLS to be 18 nm and
increased to 23 nm after antibody conjugation as shown in
Figure 1C. For PF-TC6FQ Pdots, the average diameter
increased from 17 nm to 22 nm after antibody
functionalization. The size increases could be attributed to the
surface functionalization of PSA antibody (~29 kDa) as
revealed in Figure 1D. We further executed gel electrophoresis
to examine their surface charge properties and also measured
their corresponding zeta potentials. The results in Figure 1E
suggest that the antibody functionalization led to the slower
electrophoretic mobility of the nanoparticles due to the
reduction in the negative zeta potentials as well as the
increased diameters of nanoparticles. The zeta potentials of
PCA nanoparticles and PF-TC6FQ Pdots reduced from -34
mV to -26 mV and from -43mV to -28 mV, respectively.
These results indicate the successful antibody conjugation on
the nanoparticle/Pdot surfaces.
test zone to form sandwich-shaped immune complexes. In this
scenario, FRET occurred between the PCA nanoparticles and
the PF-TC6FQ Pdots, generating the emission color transition
from sky blue to orange red. As a result, the quenching of the
PCA fluorescence and the enhancement of the PF-TC6FQ
emission could be observed simultaneously. On the other hand,
the absence of the PSA target biomarker led to a negative
signal, in which no FRET occurred on the test line and thus
the fluorescence of PCA nanoparticles remained unchanged.
The first control was fabricated with bare PCA nanoparticles
to serve as a reference of no FRET. The second control line
was modified with IgG antibodies to selectively bind with
PSA-functionalized PF-TC6FQ Pdots and then served as
another reference of FRET. The detection results could be
quickly interpreted by naked eyes under a portable 410 nm
flashlight. More precise and quantitative analysis of the target
concentration can be performed by measuring the emission
ratios of the test line to the control line(s) (T/C1 or T/C2).
We first assessed the targeting specificity of these probes in
ICTS. As shown in Figure 2A, the emission color on the test
zone turned from blue sky to orange red after reaction with
PSA proteins, indicating the occurrence of FRET phenomenon
after the chelation of PCA with PF-TC6FQ. For the other
samples, the emission of the test lines remained almost
unchanged, suggesting negligible or minimal nonspecific
protein adsorption and interference. Because the emission
profile of PCA nanoparticles overlapped partially with that of
PF-TC6FQ as shown in Figure 1B, we used filters to better
observe the optical contrast. As displayed in Figure 2B, a
band-pass filter of 470 ± 25 nm was employed to obtain the
fluorescence from PCA nanoparticles alone. For the PSA
sample, the fluorescence quenching of the test line could be
seen as compared to that of the control line. The contrast,
however, is not high enough for precise quantitative analysis
due to the strong fluorescence background from the
nitrocellulose membrane (Figure S1). On the contrary, the use
of a long-pass filter of 600 nm produced a much higher
contrast as shown in Figure 2C. These results indicate that the
qualitative fast screening could be achieved by the emission
color shift while the quantitative interpretation could be
Detection Mechanism of FRET-Based ICTS. Once we
successfully conjugated the PSA antibodies onto the surfaces
of the nanoparticles, we can assemble these materials together
in
a lateral-flow test strip to evaluate their sensing
performance. As depicted in Scheme 2, the conjugate pad was
loaded with PSA (detection)-modified PF-TC6FQ Pdots. The
microporous nitrocellulose membrane was fabricated with one
test line (T) and two control lines (C1 and C2) for optimal
comparison. For the test line, we tailored with PSA (capture)-
functionalized PCA nanoparticles. For the control lines, the
first one (C1) was made by bare PCA nanoparticles and the
other one (C2) was modified with IgG secondary antibodies.
Upon the addition of the blood samples onto the sample port,
the blood cells were selectively blocked by the porous
membrane and only the plasma/serum could migrate through
the test strip via capillary flow. This means that this ICTS can
be readily used for whole blood samples without pretreatment
because the interferences such as red blood cells will be
filtered out during migration. In the presence of PSA antigens,
PF-TC6FQ Pdot-antibody conjugates were anchored on the
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realized based on fluorescence ratio of T/C2. The reaction
onto the conjugate pad. From the images as exhibited in
time for this assay was set to 10 min because the T/C2 ratio
reached a plateau at 10 min of running time (Figure 2D). It is
worth mentioning that it took about 2 min for the sample to
flow from sample pad to absorbent pad to have a detectable
signal. The statistical analysis of the test strips (Figure 2E-F)
again confirmed that the quantitative analysis could be
performed based on emission ratios of T/C2.
Figure 3A, it is obvious to find that the emission color of the
test line turned from sky blue to intermediate yellow then red
as the concentrations of PSA increased from 0 ng/mL to 12
ng/mL. The transition point fell between 4 ng/mL and 6
ng/mL, where the emission color transferred from yellow to
red. This point is beneficial for rapid screening because PSA
levels of over 4 ng/mL are recommended to proceed with a
prostate biopsy for clear diagnosis of prostate cancer. The
traffic-light signals were successfully realized by the FRET
strategy in which the emission color on the test zone
corresponded to the PSA level. The blue fluorescence on the
test zone indicated a low PSA level (negligible FRET), while
the appearance of yellow emission on the test line represented
a middle PSA level (moderate FRET). The emergence of red
emission on the test line, however, suggested a higher PSA
level than the normal condition. This methodology highly
enhanced the discriminability of the test results by directly
recognizing the fluorescence color on the test line without the
requirement of measuring its absolute fluorescence intensity
like traditional ICTS. To further perform the quantitative
analysis of PSA concentrations by this FRET-based ICTS, we
used a 600-nm long-pass filter to selectively observe the
fluorescence signal from PF-TC6FQ Pdots (Figure 3B)
because the intensity of PF-TC6FQ is directly proportional to
the concentration of PSA. The correlation between the
emission intensities of T/C2 and PSA quantities was plotted in
Figure 3C. A dynamic range from 2 to 10 ng/mL was
displayed in the inset of Figure 3C. The limit of detection can
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ng/ml
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ng/ml
ng/ml
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C1
T
B
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be estimated by 3/m,
in which  represents the
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the calibration curve. The detection limit of PSA determined
by this FRET-based ICTS platform is 0.32 ng/mL.
0
.6 0.0
0
.2
0.4
0.6
0.8
1.0
Log [PSA] ng/mL
.4
.2
.0
Table 1. Performance Evaluation of the FRET-Based Immunoassays in
PSA-Spiked Blood.
[PSA]
[PSA]
analyte
PSA
nominal
(ng/mL)
determined±SD
(ng/mL)
Recovery (%) CV (%)
1
10
[
PSA] ng/mL
2.00
4.00
6.00
8.00
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3.82 ± 0.13
5.85 ± 0.15
7.96 ± 0.16
9.77 ± 0.21
102
95
6.34
3.40
2.56
2.01
2.14
Figure 3. Quantitative analysis of PSA concentrations with
FRET-based platforms. (A) Photographic images of ICTS under
98
100
98
4
10 nm UV excitation upon detection of analytes containing 0-12
1
0.00
ng/mL PSA antigens. (B) Their corresponding fluorescence
photographs excited using a 410 nm lamp with a 600-nm long-
pass filter. (C) The fluorescence intensity ratios of T/C2 with a
long-pass filter of 600 nm. The upper-left corner inset exhibits the
calibration curve with PSA concentrations ranging from 2 to 10
ng/mL. The error bars in C show standard deviations obtained
from over 5 replicates.
Measurement of PSA in Real Whole Blood Analytes. To
further evaluate the practicality of this FRET-based ICTS in
real human blood, we prepared fresh blood samples (from
healthy volunteers) containing PSA by spiking known amount
of PSA inside. The test results were summarized in Table 1 in
which the number of the analytes is 5 for each concentration.
The detection results exhibited the good agreement with the
spiked values, indicating the high reliability of this platform. It
is worth mentioning that the whole blood samples were
directly used and the sample pretreatment was not required in
this ICTS platform. This could highly eliminate the gross error
in most ICTS systems. The test procedures were demonstrated
in Video S1, where only one drop of blood was required for
the test. The recovery values were all above 95% with CV of
ca. 2-6 %. All of the aforementioned results suggest the
facileness and potential of this FRET-based immunoassay for
use in clinical analytes.
Quantitative Analysis of PSA Biomarkers in Simulated
Physiological Samples. The merit of the FRET-created
traffic-light signals is that the emission color of the test line is
directly relative to the target concentrations and can be easily
recognized by the naked eye. The quantitative determination
of the analyte concentrations could be further realized by
measuring the emission intensity ratios of T/C. The simulated
real samples we used here contain 0-30 L of PSA (0.1 ng/L),
3
2 L of 10% PEG, 48 L of 20 mM HEPES, and 10 % of
human serum. The spiking of human serum helped simulate
real analytes and could also reduce the nonspecific protein
binding. The role of PEG is the same with human serum while
it could at the same time prevent the sticking of Pdot probes
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Supporting Information.
A
B
:
CEA antigen
This material is available free of charge via the Internet at DOI:
Supplementary data and NMR spectra.
:PSA antigen
Testing processes in whole blood analytes.
AUTHOR INFORMATION
T1(CEA) T2(PSA)
C
C
Corresponding Author
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PSA:0ng/mL
CEA:0ng/mL
PSA:10ng/mL PSA:0ng/mL PSA:10ng/mL
CEA: 0ng/mL CEA:5ng/mL CEA: 5ng/mL
PSA:0ng/mL
CEA:0ng/mL
PSA:10ng/mL PSA:0ng/mL
CEA: 0ng/mL CEA:5ng/mL CEA: 5ng/mL
PSA:10ng/mL
*E-mail: yhchan@nctu.edu.tw
Notes
C
T2
T1
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We would like to thank the MOST, Taiwan (grant No. 108-2113-
M-009-023-MY3) and National Chiao Tung University for the
research support.
Figure 4. (A) Schematic illustrating the preparation of FRET-
based ICTS with multiplexing sensing ability. (B) Photographic
images of the test strips upon detection of analytes containing one
or two tumor biomarker(s) under 410 nm light. (C) Their
corresponding images with a long-pass filter of 600 nm.
REFERENCES
(
1) Gubala, V.; Harris, L. F.; Ricco, A. J.; Tan, M. X.; Williams, D. E.
Point of Care Diagnostics: Status and Future. Anal. Chem. 2012, 84,
487-515.
Multiplexed Detection of PSA and CEA. One of the
advantages of this FRET-based ICTS is its capability for
detecting multiple targets in a single test strip. The fabrication
of the test strips with multiplexing ability was illustrated in
Figure 4A. In the test strip, we reserved only one control line
made of bare PCA nanoparticles for color comparison. For the
first test line (T1), we modified with carcinoembryonic
antigen antibody (CEA, 10-7883)-functionalized PCA
nanoparticles. The second test line was remined to be the
PSA-functionalized PCA nanoparticles. Two conjugate pads
consisting of PF-TC6FQ-CEA and PF-TC6FQ-PSA
conjugates were stacked on top of each other. We first assess
the specificity of this multiplexed ICTS, as exhibited in Figure
4B-C. It is obvious to find out that only the corresponding test
line appeared to have FRET in the presence of the target
antigen, indicating negligible cross reactivity of this test strip.
We intentionally used a lower concentration of CEA (5 ng/mL)
as compared to PSA (10 ng/mL) and were excited to found
that the emission color discrepancy between two test lines
could be distinguished (Figure 4B). With the aid of a long-
pass filter, the fluorescence intensities from PF-TC6FQ Pdots
could be easily quantified. This design is suitable for screening
multiple targets simultaneously and provides the intuitive
signals for health conditions. The above results demonstrated
that the FRET-created traffic light ICTS has great potential for
further development of POC systems in healthcare monitoring.
(
2) Nayak, S.; Blumenfeld, N. R.; Laksanasopin, T.; Sia, S. K. Point-
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In summary, we developed a brand-new type of ICTS based on
FRET-created signals for intuitive qualitative diagnosis and
accurate quantitative detection of PSA biomarker. Each individual
test requires only 1 drop (5-10 L) of the whole blood sample
without sample pretreatment and the test result can be interpreted
in 10 min. Moreover, the limit of detection for the PSA antigen is
about one order of magnitude lower than traditional fluorometric
immunoassay systems. More importantly, multiplexed detection
of tumor markers was successfully achieved by taking advantage
of the high FRET efficiency. We anticipate this FRET-based
methodology to have wide adoption in future generations of ICTS
platforms.
5
836-5853.
(
12) Arts, R.; Hartog, I. d.; Zijlema, S. E.; Thijssen, V.; Beelen, S. H.
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