Near-Infrared Neodymium Tag for Quantifying Targeted Biomarker and Counting Its Host Circulating Tumor Cells

Article abstract of DOI:10.1021/acs.analchem.7b02016

Quantitative information on a targeted analyte in a complex biological system is the most basic premise for understanding its involved mechanisms, and thus precise diagnosis of a disease if it is a so-called biomarker. Here, we designed and synthesized a

Full text of DOI:10.1021/acs.analchem.7b02016

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Article
An NIR Neodymium-Tag for Quantifying Targeted
Biomarker and Counting Its Host Circulating Tumor Cells
Chunlan Liu, Shu Lu, Limin Yang, Peijie Chen, Peiming Bai, and Qiuquan Wang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02016 • Publication Date (Web): 26 Jul 2017
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Analytical Chemistry
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An NIR Neodymium-Tag for Quantifying Targeted Biomarker and
Counting Its Host Circulating Tumor Cells
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†
‡
†
‡
*,‡
Chunlan Liu, Shu Lu, Limin Yang, Peijie Chen, Peiming Bai, Qiuquan Wang
*,†,§
†
Department of Chemistry & the MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, College of Chem-
istry and Chemical Engineering, Xiamen University, Xiamen 361005, China
‡
Department of Urology, Zhongshan Hospital, Xiamen University, Xiamen 361004, China
State Key Lab of Marine Environmental Science, Xiamen University, Xiamen 361005, China
§
Fax no.: +86 592 2187400; e-mail: qqwang@xmu.edu.cn; baipeiming@xmu.edu.cn
ABSTRACT: Quantitative information of targeted analyte in a complex biological system is the most basic premise for under-
standing its involved mechanisms, and thus precise diagnosis of a disease if it is a so-called biomarker. Here, we designed and syn-
thesized a neodymium (Nd)-cored tag [1,4,7,10-tetraazacyclododecane-1,4,7-trisacetic acid (DOTA)-Nd complex together with a
light-harvesting antenna aminofluorescein (AMF, λex/em = 494/520 nm), AMF-DOTA-Nd] with duplex signals, second near-infrared
1
42
(
NIR) window luminescence (λem = 1065 nm, 2.5 ꢀs) and stable isotopic mass ( Nd). AMF-DOTA-Nd covalently linked with an
urea-based peptidomimetic targeting group, 2-[3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid (DUPA)-8-Aoc-Phe-Phe-Cys
DUPAaFFC) (DUPAaFFC-AMF-DOTA-Nd), allowing us to detect and quantify prostate-specific membrane antigen (PSMA)
(
and its splice variants (total PSMA, tPSMA), which was set as an example of targeted biomarkers in this study, using NIR and
ICPMS with the LOD (3σ) of 0.3 ng/mL. When it was applied to the analysis of 80 blood samples from prostate cancer (PCa) and
benign prostatic hyperplasia (BPH) patients as well as healthy volunteers, we found that 320 and 600 ng/mL tPSMA could be rec-
ommended as the threshold values to differentiate BPH from PCa and for the diagnosis of PCa. Moreover, PSMA-positive CTCs
were counted using ICPMS being from 134 to 773 CTCs in the PCa blood samples of the Gleason score from 6 to 9 when the cell
membrane-spanning mPSMA was tagged. Such a methodology developed could be expected to be applicable to other clinic-
meaningful biomolecules and their host CTCs in liquid biopsy, when other specific targeting groups are modified to the NIR Nd-tag.
Today organic fluorophores, rather than the traditional radi-
oactive isotopes, are more widely used for detecting and track-
ing biomolecules and cells owing to their spatial and temporal
peak ratio of respective emission profile has to be performed
to solve this problem using, for example, the cocktails of
3
+
3+
Tb and Eu complexes because of their different optical but
1-3
13,14
resolution and nonradioactive optical properties as well.
nearly identical physicochemical behavior in solution.
Even so, we know that they frequently encounter the draw-
backs such as narrow stokes shift between excitation and
emission wavelengths, unexpected photostability (such as
photobleaching and quenching), in addition to the possible
optical interferences of self-fluorescence and scattering from
On the other hand, the nonradioactive Ln can be precisely
quantified in a matrix-independent way using inductively cou-
pled plasma mass spectrometry (ICPMS) with the features of
high sensitivity (down to ppt level) and element/isotope-
specific subunit mass-resolved selectivity as well as isotope-
dilution based accuracy. Integration of an Ln-cored complex
and a targeting ‘warhead’ (such as small molecular inhibitor
and/or homing peptide as well as antibody) should be expected
to provide duplex signals (luminescence and element/isotope-
specific mass) and, especially the quantitative information of
targeted biomolecules can be obtained via accurate ICPMS
determination of the element/isotope tagged in a conventional
liquid biopsy, even their host circulating tumor cells (CTCs)
when the biomolecules are cell membrane-spanning proteins.
We thus designed and fabricated such a reporter consisting of
an neodymium (Nd)-cored 1,4,7,10-tetraazacyclododecane-
4,5
the extremely intricate biomatrix in the UV-visible region.
Fortunately, bioanalysis based on the luminescent lanthanides
Ln), which can be sensitized by energy transfer from the out-
(
fitted light-harvesting antenna, benefits from bigger ‘stokes
shift’ to the spectral range of near-infrared (NIR) starting for-
mally from first NIR window 650-950 nm through second
NIR window 950-1400 nm and even extending up to 2500
6
,7
nm. The NIR luminescence can not only permeate into the
body without intense absorbance and scattering by biological
matrix, but also avoid the self-fluorescence background in the
UV-visible region; and the long excited emitting lifetime fea-
8
,9
tures the time-resolved detection possible. Such advantages
do make Ln-centered probes being superior optical reporters
compared to their organic analogues in bioanalysis, yet hard to
be quantitative, only providing qualitative trends in signal
intensity change since precise concentration of the reporter
must be known and, symmetry of the ligand sphere of the co-
1,4,7-trisacetic acid (DOTA) complex together with a light-
harvesting aminofluorescein (AMF) antenna moiety (AMF-
DOTA-Nd) (1 in Figure 1). Moreover, prostate-specific mem-
1
5,16
brane antigen (PSMA), a prostate cancer biomarker,
as an example of targeted biomarkers in this study. PSMA is a
00 kDa type II transmembrane glycoprotein expressed in
is set
1
10-
ordinatively unsaturated Ln-complexes must be maintained.
2
prostate epithelial cells with an internal portion of 19 amino
acids (AAs), a transmembrane portion of 24 AAs, and a 707
1
These are difficult to know and control in a very complicat-
ed biosystem. A ratiometric measurement by modulating the
17
AAs
external
portion.
ACS Paragon Plus Environment

Analytical Chemistry
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Figure 1. Structure of PSMA-targeting DUPAaFFC-AMF-DOTA-Nd tag. The antenna moiety AMF absorbs exciting light (494 nm), and
then transfer the energy to DOTA-Nd, emitting NIR light (841 nm, 1065 nm and 1350 nm). In addition, the core Nd can be accurately
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quantified using ICPMS to obtain the mass spectrometric signals with its isotopic distribution of Nd (27.2 %), Nd (12.2 %), Nd
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(23.8 %), Nd (8.3 %), Nd (17.2 %), Nd (5.7 %) and Nd (5.6 %). Peptidomemitic DUPAaFFC can specifically target PSMA.
The external portion can be specifically targeted by a urea-
1
boratories, Inc. (Andover, MA, USA). Trifluoroacetic acid
(TFA), acetonitrile (ACN) and methanol used as the compo-
nents of chromatographic mobile phases were obtained from
Sigma-Aldrich (St. Louis, MO). Recombination human PSMA
(PSMA) was obtained from R&D Systems Inc. (Minneapolis,
USA). Prostate cancer cell lines, LNCaP (lymphatic metastatic
prostatic adenocarcinoma, PSMA-positive), DU145 (brain
metastatic of prostatic adenocarcinoma, PSMA-negative) and
PC-3 (bone metastatic prostatic adenocarcinoma, PSMA-
negative) were obtained from the Cell Line Bank of the
Shanghai Institute for Biological Science (China). RPMI-1640
culture fluid, fetal bovine serum, penicillin and streptomycin
used for cell culture were obtained from Gibco (Waltham,
USA). Dialysis membranes (molecular weight cut off, 8 kDa)
were purchased from Sangon Biotech Co., Ltd. (Shanghai,
China). The blood samples were obtained from the Affiliated
Zhongshan Hospital of Xiamen University. They include 45
blood samples from PCa patients, 20 benign prostatic hyper-
plasia (BPH) patients, 8 men and 7 women healthy volunteers
(detailed information were listed in Table S1). All the blood
samples were stored at -80 °C until use. These obtained sam-
ples were approved by the ethics committee, and the patients
signed the informed consents.
8
based peptidomimetic inhibitor,
dicarboxypropyl)-ureido]pentanedioic acid (DUPA)-X (X de-
such as 2-[3-(1,3-
1
9,20
notes a peptide spacer).
Here, we synthesized DUPA-8-
Aoc-Phe-Phe-Cys (DUPAaFFC; aFFC denotes 8-Aoc-Phe-
Phe-Cys) (2 in Figure 1), and covalently linked it to AMF-
DOTA-Nd to obtain a nonradioactive NIR- and Nd-integrated
DUPAaFFC-AMF-DOTA-Nd tag (3 in Figure 1), targeting
PSMA and PSMA-positive CTCs in blood in order to demon-
strate the feasibility of the specific and duplex quantitative
bioanalysis proposed.
Experimental Section
Materials and Instrumentations. All chemicals used in
this study are of analytical grade or better without further puri-
fication. The ultrapure water used was prepared with a Milli-Q
system (Millipore Filter Co., Bedford, MA). 3-
maleimidopropionic acid, N-Boc-1,3-propane diamine, 5-
aminefluorescein, N,N-diisopropylethylamine (DIPEA), O-(7-
azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
afluorophosphate (HATU), 10 % Pd/C and chloroacetyl chlo-
hex-
ride (ClCH COCl) were bought from J&K Scientific Ltd. (Bei-
2
jing,
China);
1,4,7-tri(t-butyl
acetate)
1,4,7,10-
An ESI ion trap mass spectrometer (ESI-IT-MS) (Esquire-
LC, Bruker Daltonics, Bremen, Germany) was used for meas-
uring molecular weight and chemical composition of the syn-
thesized molecules. The operational parameters were as fol-
lows: nebulizer, 20 psi; dry gas, 8 L/min; dry temperature,
300 °C; capillary voltage, 3.0 KV. HPLC analysis were per-
formed using an Agilent 1100 series chromatographic system
tetraazacyclododecane (DO3A-tBu ester) was purchased from
Macrocyclics (Dallas, TX); L-glutamate di-tert-butyl ester
and H-L-Glu(OBn)-OtBu used for building the DUPAaFFC-
AMF-DOTA were purchased from Maya Reagent Inc.
(Zhejiang, China). Triphosgene, neodymium oxide (Nd O ,
purity > 99.9 %) and silica gel (300-400 mesh) were obtained
2 3
154
from Aladdin Reagent Inc. (Shanghai, China); Sm-enriched
(
Agilent Technologies, Palo Alto, CA) equipped with a
G1365B-MWD UV detector (190 - 600 nm) and a ZORBAX
ACS Paragon Plus Environment
Sm O (> 99 %) was purchased from Cambridge Isotope La-
2
3

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Analytical Chemistry
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00SB-C18 column (2.1 I.D. × 100 mm in length, 3.5 µm par-
ticle size). Preparative HPLC purification of the compounds
synthesized was performed using a Shimadzu 10AD HPLC
system with an SPD-M10Avp detector and an Elite Hypersil
BDS C18 column (10.0 I.D. × 250 mm in length, 5 µm particle
size). Silica gel column chromatography for purification of the
compounds was performed using silica gel (300-400 mesh) in
a 3 I.D × 150 cm in length glass column (Beijing Synthware
Glass, China). NMR spectrum was acquired on a Varian
Gemin-400 NMR instrument (400 MHz). Hitachi U3900 spec-
trophotometer and F7000 fluorescence spectrometer, Edin-
burgh FLS980 spectrometer (200 to 2500 nm) and Zeiss
LSM780 laser confocal scanning microscope (LCSM) (350 to
(a), Phe (F), and Cys (C) as starting materials. Subsequently,
the typical Michael addition between the sulfhydryl in
DUPAaFFC and maleimido group in MMA-AMF-DOTA was
subjected to react for 2 h in ammonium acetate buffer (pH 6.4)
at 37 °C, resulting in DUPAaFFC-AMF-DOTA (97 % yield).
Finally, we obtained DUPAaFFC-AMF-DOTA-Nd (3 in Fig-
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3+
ure 1) after coordination of Nd into DUPAaFFC-AMF-
DOTA. The stability constant (log K) of DOTA-Nd is high up
to 23, ensuring the stability of Nd in DUPAaFFC-AMF-
DOTA-Nd during labeling PSMA process. All the compounds
synthesized were characterized using HPLC/ESI-IT-MS and
HNMR after purification using the preparative HPLC or silica
gel column chromatography (see Figures S1-S19 and the de-
tailed procedures in the Supporting Information).
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00 nm) were used for measuring UV-visible absorption spec-
trum, fluorescence spectrum, near infrared luminescence spec-
trum, fluorescence lifetime and cell imaging. Beckman Coulter
FC500 was used for counting cells. For stable Nd isotope
analysis, an ELAN DRC II ICPMS (PerkinElmer, SCIEX,
Canada) equipped with a concentric pneumatic nebulizer and a
cyclonic spray chamber. The ICPMS operational parameters
were as follows: nebulizer Ar gas, 0.88 L/min; auxiliary Ar gas,
1.0 L/min; plasma Ar gas, 15 L/min; RF power, 1200 W; dwell
time, 100 ms; lens voltage, 7.2 V. Parameters such as Ar nebu-
lizer gas flow and lens voltage were optimized daily to obtain
the best sensitivity with ELAN DRC II Setup/Stab/Masscal
Solution. It was coupled to size exclusion chromatography
Total PSMA (tPSMA), plasmic PSMA (pPSMA) and
membrane-spanning PSMA (mPSMA) measurements. Pri-
or to quantification of PSMA in blood, the samples were ap-
propriately diluted with PBS buffer. tPSMA in the blood sam-
ple (1 mL) was determined using ICPMS and NIR after 3 h
incubation at 37 °C with DUPAaFFC-AMF-DOTA-Nd (2 µL,
5
mM) and dialyzing out the excess free DUPAaFFC-AMF-
DOTA-Nd. After the blood sample was subjected to centrifuge
2630 g, 5 min) to separate the blood plasma and cells, pPS-
(
MA and mPSMA were determined in the plasma and the re-
sidual cells.
Cell labeling and counting. The three kinds of prostate
cancer cell lines, LNCaP (PSMA-positive), PC-3 (PSMA-
(SEC) (SEC-ICPMS) in order to confirm the specificity and
stoichiometry of DUPAaFFC-AMF-DOTA-Nd toward PSMA,
23
negative) and DU145 (PSMA-negative), which were cultured
in RPMI-1640 medium containing 10% heat-inactivated fetal
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in which Superdex 75 10/300 GL column was used and Nd
was monitored. We also investigated the interactions between
DUPAaFFC-AMF-DOTA-Nd and BSA (66 kDa, Sigma)
and/or cytochrome C (13 kDa, Sigma) using SEC-ICPMS.
bovine serum, 1% penicillin, and 1% streptomycin at 37 °C in
a 5 % CO : 95 % air incubator, were used to investigate the
cell specificity of DUPAaFFC-AMF-DOTA-Nd. Each kind of
2
5
cells was seeded in a 12-well plate (10 cells per well) and
Synthesis of DUPAaFFC-AMF-DOTA-Nd. The synthetic
route is illustrated in Scheme S1 in the Supporting Information.
To link AMF and DOTA, we first derivatized 5-
aminefluorescein into 5-chloromethylamidofluorescein (75 %
incubated overnight at 37 °C. After washing, 20 ꢀL 50 nM
DUPAaFFC-AMF-DOTA-Nd,
DUPAaFFC-AMF-DOTA,
AMF-DOTA-Nd and PBS buffer (blank) were added respec-
tively into the first four-wells of the 12-well plate for 3 h. At
least, triplicate such runs were performed. After washing three
times with PBS buffer, the trypsinized off-well cells in the first
yield) using ClCH COCl in acetone at room temperature for 5
2
h. It was then conjugated with DO3A-tBu ester via a typical
nucleophilic substitution for 9 h using K CO as an acid bind-
ing agent, KI as a stabilizing agent and DMF as a solvent un-
2
3
four-wells were dissolved in ultrapure concentrated HNO3
2
1
der an anhydrous anaerobic condition at 80 ºC, so as to ob-
tain AMF-DOTA-tBu (40 %). After tBu was deprotected from
AMF-DOTA-tBu using TFA, the resulted AMF-DOTA was
overnight, and appropriately diluted prior to determination
using ICPMS; the trypsinized off-well cells in the second four-
wells were dissolved in PBS buffer for direct NIR measure-
ment; the cells in the last four-wells were fixed with 4 % para-
formaldehyde for 15 min at 4 ºC and stained cell nucleus with
4',6-diamidino-2-phenylindole (DAPI, λem = 461 nm, blue), in
addition to DUPAaFFC-AMF-DOTA-Nd, DUPAaFFC-AMF-
DOTA, AMF-DOTA-Nd and PBS buffer, for imaging the cell
nucleus and mPSMA on the surface of the cells using LCSM.
To further confirming the specificity of the probe towards
PSMA-positive cancer cells, blocking studies were performed.
LNCaP cells were pre-incubated with 50 nM DUPAaFFC for
3
+
mixed with excess Nd in ammonium acetate buffer (pH 6.4)
at 37 ºC for 3 h to obtain AMF-DOTA-Nd (1 in Figure 1). 3-
maleimidopropionic acid reacted with N-Boc-1,3-propane
diamine, and then used TFA for deprotecting Boc to obtain N-
(3-aminopropyl)-3-maleimidopropanamide (56 %). It contains
both –NH and maleimido group serving as a linker between
2
AMF-DOTA-tBu and the peptidomimetic guiding moiety.
Aminolysis of AMF-DOTA-tBu using N-(3-aminopropyl)-3-
maleimidopropanamide and then deprotection of tBu resulted
in MMA-AMF-DOTA (14 %). In parallel, synthesis of the
peptidomimetic guiding moiety started from L-glutamate di-
tBu to react with triphosgene for 2 h using triethylamine (TEA)
as an acid binding agent at -78 °C under anhydrous anaerobic
condition, and then a solution of H-L-Glu(OBn)-OtBu together
with TEA in DCM was added to react at room temperature
overnight to synthesize 2-[3-(3-benzyloxycarbonyl-1-tert-
butoxycarbonyl-propyl)-ureido]pentanedioic acid ditert-butyl
ester (77 %). After deprotecting benzyl group using 10 % Pd/C,
3
h prior to the addition of 50 nM DUPAaFFC-AMF-DOTA-
Nd and/or DUPAaFFC-AMF-DOTA. Moreover, for counting
PSMA-positive LNCaP cells using ICPMS and NIR, 20 ꢀL 50
nM DUPAaFFC-AMF-DOTA-Nd was added to a series
known number of LNCaP cells. After 3 h incubation, washing
three times with PBS buffer to get rid of the excess free
DUPAaFFC-AMF-DOTA-Nd prior to measurements.
we
obtained
2-[3-(1,3-bis-tert-butoxycarbonyl-propyl)-
Results and discussion
ureido]pentanedioic acid-tert-butyl ester (DUPA-tBu) (68 %).
The peptidomimetic DUPAaFFC (2 in Figure 1) was obtained
through solid phase peptide synthesis using DUPA-tBu, 8-Aoc
ACS Paragon Plus Environment

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Spectroscopic characteristics of DUPAaFFC-AMF-
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DOTA-Nd. In order to evaluate the NIR luminescent and mass
spectroscopic properties of DUPAaFFC-AMF-DOTA-Nd, we
first investigated the optical properties of DUPAaFFC-AMF-
DOTA-Nd, DUPAaFFC-AMF-DOTA and AMF as well as
Nd(NO ) . AMF, DUPAaFFC-AMF-DOTA and DUPAaFFC-
3
3
AMF-DOTA-Nd had the same absorption peak at 494 nm
Figure S20). Under the excitation of 494 nm, AMF and
(
DUPAaFFC-AMF-DOTA emitted the same fluorescence peak
at 520 nm from AMF with the lifetime of 5.7 ns as expected;
while the NIR luminescence of DUPAaFFC-AMF-DOTA-Nd
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was observed at 841 nm ( F → I ), 1065 nm ( F → I11/2)
3/2
24,25
9/2
3/2
4
4
and 1350 nm ( F → I13/2),
with the longer lifetime of 2.5
3/2
ꢀs and much bigger ‘stokes shift’ of 347 nm, 571 nm and 856
nm (Figure 2), suggesting the successful energy transfer from
AMF to DOTA-Nd. In the case of Nd(NO ) , neither noticea-
3
3
ble absorption at 494 nm nor luminescence from visible to NIR
could be recorded due to the very low molar absorption coeffi-
3
cient of Nd arising from the parity-forbidden transition of f-
+
Figure 2. Emission spectra of Nd(NO ) (green line), AMF (black
3 3
2
orbital electron and its relatively small diameter. All these
6
line), DUPAaFFC-AMF-DOTA (blue line), DUPAaFFC-AMF-
DOTA-Nd (red line) of 10.0 ꢀM each in 20 mM PBS buffer (pH
7.4). Two-step scanning from 450 nm to 800 nm (visible region)
and then 800 nm to 1500 nm (NIR region) was performed under
the excitation of 494 nm.
results confirmed the crucial role of AMF for the NIR emis-
3
+
sion from Nd in DUPAaFFC-AMF-DOTA-Nd. It absorbs
the matched energy with parity-allowed transition at the triplet
state, which transformed from the singlet state of the excited
AMF via intersystem crossing. We then measured a series of
DUPAaFFC-AMF-DOTA-Nd solutions of different concen-
trations from 0 to 257.5 nM (corresponding to 0.5 µg/mL) to
make a calibration curve at 1065 nm considering that it is
strongest among the three NIR luminescence (Figure 3). The
linear dynamic range was from 0.1 to 103 nM (corresponding
2
to 0.2 to 200 ng/mL) (correlation coefficient R = 0.992), and
the limit of detection (LOD, 3σ) reached 77.2 pM (correspond-
ing to 0.15 ng/mL) with an RSD of 1.4 % at 76.4 ng/mL (n =
5). Subsequently, the mass spectroscopic property of
DUPAaFFC-AMF-DOTA-Nd was investigated using ICPMS
142
154
monitoring
Nd and,
Sm as species-unspecific isotopic
internal standard. Nd has much lower first ionization potential
(ca. 5.5 eV) compared with that (15.8 eV) of Ar, which is used
as the plasma gas in ICP, resulting in almost 100 % ionization
efficiency and thus very low LOD on an conventional Ar-
27
based ICPMS. The obtained results indicated that
DUPAaFFC-AMF-DOTA-Nd and Nd(NO3)3 had the same
mass spectrometric signal intensity at equal Nd concentration
Figure 3. ICPMS and NIR responses from a series of Nd(NO3)3
(black solid square for ICPMS, red solid circle for NIR) and
DUPAaFFC-AMF-DOTA-Nd (black open square for ICPMS, red
open circle for NIR) solutions (0 - 250 nM), respectively.
(
sponse due to ICP is a very hard ionization source. The LODs
Figure 3), demonstrating a species-independent ICPMS re-
1
42
(
3σ), when monitoring Nd (its abundance is 27.2 %, the
factor for achieving a specific quantification of PSMA and count-
0-32
3
highest one among Nd isotopes), was down to 2.8 pM (corre-
sponding to 5.4 pg/mL), almost 28-fold lower than that of the
NIR measurement; and the linear dynamic range was up to
ing its host CTCs. According to the previous studies,
DUPA-
X-Dap-Asp-Cys (Dap denotes β-L-diaminopropionic acid) inter-
acts with the external domain of PSMA molecule with 1:1 stoichi-
19,33
2
5
57.5 nM DUPAaFFC-AMF-DOTA-Nd (corresponding to
2
00 ng/mL; higher concentrations were not tested) (R = 0.997)
ometry with high affinity (K = 14 nM).
d
The carbonyl oxygen
of Glu-C(O)- Glu in DUPA was found to coordinate directly with
the zinc in the active site located at the bottom of S1 pocket do-
main of PSMA, the α and α′-carboxyl interacting with Arg210 and
Arg534, and the γ-carboxyl forming a third salt bridge with
Lys699 as autodocked in Figure S21. Moreover, additional hydro-
phobic and hydrogen bonding interactions from the two Phe and
ureal nitrogen in DUPA-8-Aco-Phe-Phe-Dap-Asp-Cys with S1
pocket were proved to reinforce the affinity of DUPA-8-Aco-Phe-
with an RSD of 0.5 % at 10.4 ng/mL (n = 7). All the results
suggested that both the NIR luminescence and ICPMS signals
could be obtained from the same Nd-cored DUPAaFFC-AMF-
DOTA-Nd. Compared with the trifunctional probe (λ
=
494/520 nm; Eu) that we previously designed and synthe-
ex/em
153
28
sized, in which AMF and DOTA-Eu worked separately; and
the AuNP-quenched activity-released Eu-BCTOT-peptide
19
Phe-Dap-Asp-Cys to PSMA. In order to prove the labeling effi-
1
53
probe (λex/em = 350/614 nm; Eu) that worked by the media-
tion of a protease, DUPAaFFC-AFM-DOTA-Nd is an Nd-
ciency of DUPAaFFC-AMF-DOTA-Nd tag, which was designed
and synthesized in this study, toward PSMA, we used SEC that is
able to separate the molecules of different weights and/or sizes,
2
9
cored second NIR- and mass-integrated duplex tag (λ
=
ex/em
142
94/1065 nm; Nd).
142
4
together with ICPMS for monitoring Nd. The results obtained
Figure 4) indicated that a new peak appeared at 9.5 min in the
case of mixing PSMA (5 nM) with DUPAaFFC-AMF-DOTA-Nd
15 nM) in 100 ꢀL 50 mM PBS buffer (pH 7.4), in addition to
(
Selectivity of DUPAaFFC-AMF-DOTA-Nd toward
PSMA. In addition to the readout properties, the targeting
feature of DUPAaFFC-AMF-DOTA-Nd is another important
(
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Analytical Chemistry
cond NIR window microscopy at hand this moment (although
the peak at 19.4 min compared with DUPAaFFC-AMF-
1
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DOTA-Nd (15 nM) alone. The new peak was PSMA-
DUPAaFFC-AMF-DOTA-Nd because of its much larger mo-
lecular weight thus eluted earlier from the SEC, and the deter-
mination of Nd as well. Moreover, the peak area ratio was
2:1 (19.4 min to 9.5 min), and the sum of them equaled to that
of DUPAaFFC-AMF-DOTA-Nd alone. This observed fact
suggested that not only complete tagging of PSMA by
DUPAaFFC-AMF-DOTA-Nd, but also the stoichiometry is
PSMA-DUPAaFFC-AMF-DOTA-Nd could be determined
using an Edinburgh FLS980 fluorescence spectrophotometer at
1065 nm), DUPAaFFC-AMF-DOTA-Nd and/or DUPAaFFC-
AMF-DOTA that emits green fluorescence at 520 nm owing to
142
3+
the absence of Nd were used in parallel to quantify and visu-
alize the DUPAaFFC-targeted PSMA-positive cells using
ICP-MS and under a conventional LCSM. The results ob-
tained indicated that the PSMA-positive LNCaP cells could be
counted using ICPMS (Figure S24a ) and NIR (Figure S24a )
1
:1. Selectivity of DUPAaFFC-AMF-DOTA-Nd was also
1
2
investigated using other model proteins such as BSA and cyto-
chrome C together with PSMA. No DUPAaFFC-AMF-
DOTA-Nd tagged BSA and cytochrome C were detected, indi-
cating its specificity toward PSMA (Figure S22). In this way,
the LOD (3σ) was down to 2.9 pM PSMA (corresponding to
with DUPAaFFC-AMF-DOTA-Nd, and could also be fluores-
cently detected (Figure S24a ’) and green-visualized (Figure
0
1
2
3
4
5
6
7
8
9
0
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0
2
S24a ’) at 520 nm when using DUPAaFFC-AMF-DOTA; but
4
not when only PBS and AMF-DOTA-Nd without the targeting
DUPAaFFC were used (Figure S24a ’’-a ’’ and a ’’’-a ’’’). In
1
5
1
5
0
(
.3 ng PSMA/mL) with a linear dynamic range up to 245 nM
the cases of PSMA-negative DU145 and PC-3, either
DUPAaFFC-AMF-DOTA-Nd and DUPAaFFC-AMF-DOTA
or AMF-DOTA-Nd and PBS did not give any detectable sig-
nals (Figure S24b -b , b ’-b ’, b ’’-b ’’, b ’’’-b ’’’ and c -c ,
2
corresponding to 25 µg/mL, R = 0.995; higher concentrations
were not tested) with an RSD of 1.5% at 61.1 ng/mL (n = 10)
using ICPMS. While the linear range obtained was from 0.1 to
1
5
1
5
1
5
1
5
1
5
2
98 nM (corresponding to from 10 ng/mL to 10 µg/mL, R =
c ’-c ’, c ’’-c ’’, c ’’’-c ’’’), suggesting the specificity of
1 5 1 5 1 5
0.990) (Figure S23), and the LOD (3σ) was 95 pM PSMA
DUPAaFFC-AMF-DOTA-Nd and DUPAaFFC-AMF-DOTA
toward the PSMA-positive LNCaP cell. Moreover, when we
used DUPAaFFC to block the LNCaP cell membrane-
spanning mPSMA prior to the addition of DUPAaFFC-AMF-
DOTA-Nd and/or DUPAaFFC-AMF-DOTA, neither ICPMS
and NIR luminescent and/or green fluorescent signals nor
green-visualization could be observed (Figure S24a ’’’’-a ’’’’),
(corresponding to 9.7 ng PSMA/mL) using NIR at 1065 nm
with an RSD of 3.5 % at 61.1 ng/mL (n = 5). Comparing to
NIR, the LOD of PSMA using ICPMS is almost 33 times low-
er, and the sensitivity remains almost unchanged as that of
DUPAaFFC-AMF-DOTA-Nd itself, indicating again the ma-
trix-independent advantage of ICPMS. When compared with
the sensitivities obtained from the western blot using fluores-
cent detection and ProteinChip arrays (about 1 ꢀg/mL) with
1
5
again confirming the specificity of this tag toward the PSMA-
positive LNCaP cell. Subsequently, different number of the
2
5
surface-enhanced laser desorption/ionization time-of-flight
34,35
LNCaP cells ranging from 5 × 10 to 5 × 10 that were counted
by flow cytometry was labeled with DUPAaFFC-AMF-
DOTA-Nd tag for quantifying the amount of mPSMA ex-
pressed per LNCaP cell (Figure 5). The results obtained indi-
mass spectrometry,
the LOD obtained using ICPMS in this
study is much lower; and comparable to those of fluorescence-
based dual-monoclonal sandwich assay (less than 1 ng/mL)
and electrochemical microfluidic immunoarray (0.15 pg/mL)
7
cated that one LNCaP cell expressed (3.6 ± 0.1) × 10 (ICPMS)
3
6,37
7
with additional enrichment procedures.
and (3.6 ± 0.2) × 10 (NIR) mPSMA molecules in the case that
4
5
weight of PSMA (100 kDa) into account. It should be noted
× 10 LNCaP cells were employed, taking the molecular
that ICPMS gave similar results even the cell number was 5 ×
10 , but not the case when using NIR owing to its lower detec-
2
tion limit. On the basis of the LOD of PSMA, down to 48 and
1596 LNCaP cells could be counted directly using ICPMS and
NIR together with DUPAaFFC-AMF-DOTA-Nd. Considering
the Avogadro constant saying that one mole contains 6.023 ×
23
1
0
particles, the cell-counting sensitivity theoretically
-
23
-21
reached 8.0 × 10 (ICPMS) and 2.6 × 10 (NIR) mole level.
Compared to NIR, ICPMS was superior in either the matrix-
independent m/z signals or 33 times higher sensitivity.
Figure 4. SEC-ICPMS chromatograms of DUPAaFFC-AMF-
DOTA-Nd (15 nM) (black line) and, the mixture of PSMA (5 nM)
142
with DUPAaFFC-AMF-DOTA-Nd (15 nM) (red line). Nd was
monitored; a Superdex 75 10/300 GL column (10 mm I.D. × 300
mm in length) and 50 mM ammonium bicarbonate (pH 7.4) mo-
bile phase with the flow rate of 0.8 mL/min were used.
Specificity of DUPAaFFC-AMF-DOTA-Nd toward
PSMA-positive cells. In order to examine the tagging specific-
ity of DUPAaFFC-AMF-DOTA-Nd toward cells, we used
LNCaP (PSMA-positive), DU145 (PSMA-negative) and PC-3
Figure 5. Relationship between the LNCaP cell number and the
intensity of ICPMS (black square) or NIR (red circle). Log 10
(PSMA-negative) cell lines. Because we did not have an se-
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Analytical Chemistry
Page 6 of 9
scale was used after the break (x axis break from 1 to 100, Y axis
for ICPMS from 0.01 to 0.1, for NIR from 1 to 100) at 5% of the
axis.
were 134 to 773 PSMA-positive CTCs within 20 % RSD in
the blood samples along with the increase in the Gleason score
from 6 to 9, being in accordance to the fact that CTCs are
1
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9
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43-46
within 1000 in 1 mL blood.
It is worth noting that PSM-E,
PSMA in blood samples. Before applying the method de-
veloped here to the real blood samples, we were aware that
in addition to PSMA, also contribute to mPSMA, the counted
CTCs here should be more accurate than those merely based
on the amount of PSMA expressed on the cells, considering
PSMA and PSM-E have a similar molecular weight according
PSMA has its splice variants such as PSM’, PSM-C, PSM-D
38-41
and PSM-E.
All these variants have the same DUPAaFFC
41
targeting domain, thus the determined amount in the blood
samples should be the sum of PSMA and its splice variants. It
was termed as total PSMA (tPSMA) as shown in Figure 6 (de-
tailed data are listed in Table S1). tPSMA determined in PCa
blood samples ranged from 295.4 to 2605.1 ng/mL, the mean
and median values were 800.7 ng/mL and 676.8 ng/mL. When
analyzing these data (the mean value of Gleason score 6, 569.3
ng/mL; 7, 622.7 ng/mL; 8, 730.8 ng/mL; 9, 1781.1 ng/mL), we
could see that the tPSMA not only increased along with their
Gleason score, but also were significantly higher than those of
BPH blood samples from 193.3 to 454.3 ng/mL, 317.4 ng/mL
to their mRNA. Clearly, results of counting CTCs would be
much more accurate when a more specific membrane-spanning
biomarker without the splice variants was targeted in the near
future.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
6
7
8
9
0
Table 1. The concentrations of tPSMA, pPSMA and
mPSMA, and the number of PSMA-positive CTCs in the
six selected PCa blood samples of different Gleason
a
score
tPSMA in
blood
pPSMA in
plasma
(ng/mL)
mPSMA
in cells
(ng/mL)
cell
number
(copy)
Gleason
score
(mean) and 309.0 ng/mL (median) (P < 0.001). Thus, 320
No.
Age
(
ng/mL)
ng/mL might be roughly thought as a threshold value to differ-
entiate BPH and PCa; and 600 ng/mL could be a threshold
value for diagnosis of PCa according to Gleason score 6 that is
1
2
3
4
5
6
50
75
63
60
78
83
6
6
7
7
8
9
324.6 ± 4.4
741.5 ± 7.9
323.8 ± 3.6
740.6 ± 8.5
790.1 ± 6.8
750.8 ± 7.4
1000.4 ± 7.1
2495.6 ± 14.3
0.8 ± 0.1
1.0 ± 0.2
2.2 ± 0.3
2.5 ± 0.3
3.4 ± 0.4
4.6 ± 0.4
134 ± 17
168 ± 33
367 ± 50
420 ± 50
571 ± 68
773 ± 68
42
thought to be a definite PCa. . By comparison, tPSMA in the
792.3 ± 10.1
753.4 ± 5.7
blood samples of healthy men and women were much lower,
ranging from 152.4 to 362.5 ng/mL and 30.4 to 49.3 ng/mL,
with the mean values of 231.0 and 38.9 ng/mL, and median
values of 222.4 and 40.1 ng/mL (P < 0.001).
1003.6 ± 10.2
2500.2 ± 15.2
a, each sample was measured three times.
Conclusions
We designed and synthesized an NIR luminescent (1065 nm)
142
and mass spectroscopic ( Nd) duplex AMF-DOTA-Nd tag.
When it covalently linked with a PSMA-targeting pep-
tidomimetic ‘warhead’, DUPAaFFC, DUPAaFFC-AMF-
DOTA-Nd demonstrated its ability as a specific duplex readout
probe, exemplified in the quantification of tPSMA in the PCa,
BPH and healthy blood samples using ICPMS. The tPSMA
concentration in blood can be used as an index to not only
diagnose PCa (threshold value, 600 ng/mL) but also differenti-
ate BPH from PCa (320 ng/mL); moreover, hundreds of
PSMA-positive CTCs in the PCa blood samples were counted
via mPSMA using ICPMS. If having a second NIR window
microscopy at hand, we believe that NIR-imaging of mPSMA
and its host CTCs would become reality. Future work will be
focused on the modification of the NIR Nd-tag with other tar-
geting groups to expand its application to the quantification
and counting of different biomarkers and CTCs.
Figure 6. Boxplots of tPSMA concentrations in the PCa with
different Gleason score, BPH, healthy male and female blood
samples. The boxes represent the 25th and 75th percentiles; the
whiskers represent the 5th and 95th percentiles; and outliers are
displayed by circles. The black lines represent the median value;
the red lines represent the mean value.
ASSOCIATED CONTENT
Supporting Information
Counting PSMA-positive CTCs. Among PSMA and its
splice variants, only PSMA and PSM-E are known as the
The Supporting Information is available free of charge on the
ACS Publications website.
43
transmembrane proteins anchoring on its host cells, mPSMA;
Synthesis and characterization of DUPAaFFC-AMF-DOTA-
Nd, UV-Vis absorption spectra, Autodocking, SEC-ICP-MS, cali-
bration cures of PSMA using ICPMS and NIR together with
DUPAaFFC-AMF-DOTA-Nd tag, specificity of DUPAaFFC-
AMF-DOTA-Nd toward PSMA-positive cells, results of total
PSMA in real blood samples (PDF)
while the other plasmic variants including PSM’, PSM-C and
PSM-D (termed as pPSMA here) are either inside or out of the
38
cells entering into the blood circulation. To simplify counting
the PSMA-positive CTCs in the real PCa blood samples, we
centrifuged out the plasma, where pPSMA exist. Afterwards,
we counted the PSMA-positive CTCs in the six PCa blood
samples selected (1 mL each) of different Gleason scores via
the determination of mPSMA in the blood residues containing
the blood cells using ICPMS together with DUPAaFFC-AMF-
DOTA-Nd tag (Table 1). Results obtained indicated that there
AUTHOR INFORMATION
Corresponding Author
* E-mail: qqwang@xmu.edu.cn; Fax: +86 (0)592 2187400.
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*
E-mail: baipeiming@xmu.edu.cn
(27) Wendt, K.; Gottwald, T.; Mattolat, C.; Raeder, S. Hyperfine
Interact. 2014, 227, 55–67.
28) Zhang, Z. B.; Luo, Q.; Yan, X. W.; Li, Z. X.; Luo, Y. C.;
Yang, L. M.; Zhang, B.; Chen, H. F.; Wang, Q. Q. Anal. Chem. 2012,
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ORCID
Qiuquan Wang: 0000-0002-5166-4048
8
4, 8946−8951.
29) Feng, D.; Tian, F.; Qin, W. J.; Qian, X. H. Chem. Sci. 2016, 7,
246–2250.
(
Notes
The authors declare no competing financial interest.
2
(30) Barinka, C.; Byun, Y.; Dusich, C. L.; Banerjee, S. R.; Chen,
Y.; Castanares, M.; Kozikowski, A. P.; Mease, R. C.; Pomper, M. G.;
Lubkowski, J. J. Med. Chem. 2008, 51, 7737–7743.
(31) Banerjee, S. R.; Foss, C. A.; Castanares, M.; Mease, R. C.;
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Pomper, M. G. J. Med. Chem. 2008, 51, 4504–4517.
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Chem. 2010, 53, 5333–5341.
ACKNOWLEDGMENTS
This work was financially supported by the National Natural Sci-
ence Foundation of China (Grants 21535007, 21475108,
21275120), the National Basic Research 973 Program (Grant
2014CB932004) and National Science and Technology Basic
Work (2015FY111400), as well as the Foundation for Innovative
Research Groups of the National Natural Science Foundation of
China (Grant 21521004), Program for Changjiang Scholars and
Innovative Research Team in University (PCSIRT, Grant
IRT13036).
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Figure captions
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Analytical Chemistry
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Figure 1. Structure of PSMA-targeting DUPAaFFC-AMF-DOTA-Nd tag. The antenna moiety AMF absorbs exciting light (494 nm), and
then transfer the energy to DOTA-Nd, emitting NIR light (841 nm, 1065 nm and 1350 nm). In addition, the core Nd can be accurately
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quantified using ICPMS to obtain the mass spectrometric signals with its isotopic distribution of Nd (27.2 %), Nd (12.2 %), Nd
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23.8 %), Nd (8.3 %), Nd (17.2 %), Nd (5.7 %) and Nd (5.6 %). Peptidomemitic DUPAaFFC can specifically target PSMA.
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Figure 2. Emission spectra of Nd(NO ) (green line), AMF (black line), DUPAaFFC-AMF-DOTA (blue line), DUPAaFFC-AMF-DOTA-
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Nd (red line) of 10.0 ꢀM each in 20 mM PBS buffer (pH 7.4). Two-step scanning from 450 nm to 800 nm (visible region) and then 800 nm
to 1500 nm (NIR region) was performed under the excitation of 494 nm.
Figure 3. ICPMS and NIR responses from a series of Nd(NO ) (black solid square for ICPMS, red solid circle for NIR) and DUPAaFFC-
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AMF-DOTA-Nd (black open square for ICPMS, red open circle for NIR) solutions (0 - 250 nM), respectively.
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Figure 4. SEC-ICPMS chromatograms of DUPAaFFC-AMF-DOTA-Nd (15 nM) (black line) and, the mixture of PSMA (5 nM) with
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DUPAaFFC-AMF-DOTA-Nd (15 nM) (red line). Nd was monitored; a Superdex 75 10/300 GL column (10 mm I.D. × 300 mm in
length) and 50 mM ammonium bicarbonate (pH 7.4) mobile phase with the flow rate of 0.8 mL/min were used.
Figure 5. Relationship between the LNCaP cell number and the intensity of ICPMS (black square) or NIR (red circle). Log 10 scale was
used after the break (x axis break from 1 to 100, Y axis for ICPMS from 0.01 to 0.1, for NIR from 1 to 100) at 5% of the axis.
Figure 6. Boxplots of tPSMA concentrations in the PCa with different Gleason score, BPH, healthy male and female blood samples. The
boxes represent the 25th and 75th percentiles; the whiskers represent the 5th and 95th percentiles; and outliers are displayed by circles. The
black lines represent the median value; the red lines represent the mean value
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