- Email: [email protected]
Detection of three tumor biomarkers in human lung cancer serum using single particle inductively coupled plasma mass spectrometry combined with magnetic immunoassay
Journal Pre-proof Detection of three tumor biomarkers in human lung cancer serum using single particle ICP-MS combined with magnetic immunoassay
Yupin Cao, Jinsu Feng, Lifu Tang, Guichun Mo, Weiming Mo, Biyang Deng PII:
S0584-8547(19)30479-3
DOI:
https://doi.org/10.1016/j.sab.2020.105797
Reference:
SAB 105797
To appear in:
Spectrochimica Acta Part B: Atomic Spectroscopy
Received date:
10 October 2019
Revised date:
15 February 2020
Accepted date:
17 February 2020
Please cite this article as: Y. Cao, J. Feng, L. Tang, et al., Detection of three tumor biomarkers in human lung cancer serum using single particle ICP-MS combined with magnetic immunoassay, Spectrochimica Acta Part B: Atomic Spectroscopy(2019), https://doi.org/10.1016/j.sab.2020.105797
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© 2019 Published by Elsevier.
Journal Pre-proof
Detection of three tumor biomarkers in human lung cancer serum using single particle ICP-MS combined with magnetic immunoassay Yupin Cao1,2 , Jinsu Feng1 , Lifu Tang1 , Guichun Mo1 , Weiming Mo1 , Biyang Deng1,* [email protected] 1
State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources,
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School of Chemistry and Pharmaceutical Sciences, Guangxi Normal University, Guilin 541004, China
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Guangxi University of Chinese Medicine, Nanning 530200, China
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2
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* Corresponding Author.
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Telephone: +86-773-5845726, Fax: +86-773-2120958,
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Email: [email protected] (B. Deng)
Journal Pre-proof
Abstract A new method
for simultaneously determining cytokeratin fragment antigen 21-1
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(CYFRA21-1), carcinoembryonic antigen (CEA), and carbohydrate antigen (CA15-3) was
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developed using single-particle, ICP-MS-based, magnetic immunoassay. Gold nanoparticles (AuNPs), ZnSe quantum dots (QDs), and Silver nanoparticles (AgNPs) were employed to tag
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antibodies. A highly efficient capture of CYFRA21-1, CEA, and CA15-3 was achieved using
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biotinylated, antibody-coated magnetic beads. AuNPs/ZnSe QDs/AgNPs-tagged second antibodies helped realize sensitive quantification of the target antigen using single particle ICP-MS. Under
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optimum conditions, CYFRA21-1, CEA, and CA15-3 linear concentration ranges were 0.05-50 ng mL-1 , 0.02-100 ng mL-1 , and 0.6-250 mU mL-1 . The correlation coefficients were 0.9999, 0.9987,
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and 0.9970, respectively. CYFRA21-1, CEA, and CA15-3 detection limits were 0.02 ng mL-1 , 0.006
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ng mL-1 , and 0.25 mU mL-1 , respectively. Relative standard deviations (RSD) for CYFRA21-1, CEA, and CA15-3 were 3.2%, 4.4%, and 4.2%, respectively. The method simultaneously detected
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CYFRA21-1, CEA, and CA15-3 in human lung cancer serum samples with recoveries of between 89.8% and 101%.
Keywords: CYFRA21-1; CEA; CA15-3; Single particle-ICP-MS; Magnetic immunoassay
Journal Pre-proof 1. Introduction Cancer is commonly fatal to humans [1]. Lung cancers continue as the most common, lethal cancer and account for more than 25% of all U.S. cancer deaths [2]. It is classified into two major groups: small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) [3]. Presently cytokeratin fragment antigen 21-1 (CYFRA21-1) is the preferred biomarker for detecting NSCLC, particularly squamous cell carcinoma [4]. Carcinoembryonic antigen (CEA) is an accepted
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tumor marker for colorectal cancer. It has been reported as a prognostic lung cancer marker [5]. Carbohydrate antigen (CA15-3), encoded by MUC1, which is known as a tumor marker for breast
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cancer [6]. Tumor biomarkers mirror tumor tissue, cell differentiation, and cell function. Early stage
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detection of tumor biomarkers contributes to diagnosis, classification, prognosis judgment, and
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treatment [7].
Several methods have been used to analyze biomarkers, including: enzyme-linked
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immunosorbent assay (ELISA) [8]; fluoroimmunoassay (FIA) [9]; chemiluminescent immunoassay (CLIA) [10,11]; electrochemical immunoassay (ECIA) [12,13]; and inductively coupled plasma
na
mass spectrometry (ICP-MS) [14-19]. An element-specific detector, ICP-MS is now one of the most powerful elemental analysis tools having the advantages of high sensitivity, low matrix effects, wide
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dynamic range, and multiplex detection ability [20,21]. Since the time resolved transient signals
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induced by the flash of ions arising from the ionization in the plasma torch can reflect the concentrations of nanoparticles, the concentration of nanoparticle-tagged immunocomplex could be quantified by the frequency of transient signals [18]. ICP-MS element tagging has the potential to achieve multiplex analysis for massive bioassays and clinical diagnosis [18]. Detecting one biomarker at a time is very time-consuming when measuring multiple biomarkers. The simultaneous immunoassay of multianalytes has attracted much attention. It improves assay efficiencies and lowers sample consumption [22]. Hong et al. reported on an ICP-MS-based multiple magnetic immunoassay which simultaneously detected three gynecological tumor biomarkers in clinical serum samples. A highly efficient capture of HER-2, HE4, and CA15-3 using biotinylated antibody-coated magnetic beads was obtained. Using a lanthanide ion-tagged, second antibody
Journal Pre-proof helped realize a sensitive quantification of the target antigen using ICP-MS [6]. Ko et al. reported a multiplex detection of biomarkers via a sandwich-type immunoassay for AFP, NSE, and CRP [15]. Zhang et al. reported an ICP-MS-based immunoassay using golden nanoparticles (AuNPs) and silver nanoparticles (AgNPs) to tag elements for determining CEA and AFP in human serum samples [16]. This article first reports on an ICP-MS-based, multiple immunoassay using AuNPs, AgNPs, and
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ZnSe quantum dots (QDs) to tag elements for simultaneously determining CYFRA21-1, CA15-3, and CEA in human lung cancer serum samples. First, streptavidin- functionalized magnetic beads
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were conjugated with a primary antibody to capture target proteins. Second, analyte quantification
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was realized via a sandwich-type immunoreaction using streptavidin- functionalized magnetic beads and a biotinylated antibody. AuNPs, AgNPs, and ZnSe QDs were synthesized as probes to determine
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CYFRA21-1, CA15-3, and CEA in human lung cancer serum samples via SP-ICP-MS. The
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suspension solutions were separated by a permanent magnet. Single particles were direc tly introduced into ICP-MS with a custom, gas pressure-assisted, sample introduction system which was
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successfully used on human lung cancer serum samples. Comparing to tag one element, the
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2. Experimental
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immunoassay of multi-elements can improve assay efficiency and reduce sample consumption.
2.1. Instrumentation
A NexIon 300X ICP-MS system (Perkin Elmer, USA) with a custom, gas pressure-assisted sample- introduction system (GPASIS) [23] determined
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Au,
107
Ag, and
64
Zn in a single particle
mode. The optimized ICP-MS operating parameters appear in Table 1. AuNPs, AgNPs and ZnSe QDs were characterized using Fourier transform infrared spectroscopy (FTIR, Perkin Elmer, USA), transmission electron microscope (TEM, JEM-2100, JEOL, Japan), and a Rigaku D/max 2500/pc X-ray powder diffractometer (XRD, Rigaku, Japan). Table 1
Journal Pre-proof 2.2. Reagents N-hydroxysuccinimide
(NHS,
98%),
N-(3-(dimethylamino)propyl)-N’-ethylcarbodiimide
hydrochloride (EDC, 98%) was obtained from Aladdin Industrial Corporation (Shanghai, China). Bovine serum albumin (BSA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Streptavidin functionalized magnetic beads, biotinylated monoclonal CYFRA21-1 antibody (goat, Ab1-CYFRA21-1), and CYFRA21-1 were from Guangxi Jia Ye Technology Co., Ltd (Guangxi,
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China). Rabbit anti-goat IgG antibody (Ab2-CYFRA21-1), CEA, mouse anti-CEA antibody (Ab1-CEA) and goat anti- mouse IgG antibody (Ab2-CEA), CA15-3, rabbit anti-MUC1
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(Ab1-CA15-3) and goat anti-rabbit IgG (Ab2-CA15-3) were purchased from Bioss (Beijing, China).
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Methane (99.999%) was from Dalian Special Gases (Liaoning, China). Formic acid, sodium borohydride (NaBH4 ), zinc chloride (ZnCl2 ), sodium chloride (NaCl), potassium chloride (KCl),
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di-potassium hydrogen phosphate trihydrate (K 2 HPO 4 ∙3H2 O), potassium dihydrogen phosphate
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(KH2 PO4 ) were supplied by Xilong Chemical of Guangdong (Guangdong, China). ICP-MS daily performance was checked using a multielement standard solution containing Be, Mg, In, U and Ce
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at concentrations of 1 ng mL-1 (Perkin Elmer, USA). Ultrapure water (18.2 MΩ) was used. All solutions were filtered through a 0.45 μm membrane filter before use, which supplied by Shanghai
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Xinya Purification Material Factory (Shanghai, China).
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2.3. SP-ICP-MS data processing An iterative algorithm, based on a three-time standard deviation (3σ), was used to discriminate particle pulse from the background signal for SP-ICP-MS. The determining particle events first step begins by averaging the entire data set and collecting all data points that are 3σ above the mean for the entire set. This is similar to outlier removal from a normally-distributed data set. Near-normal distribution data point distribution is assumed. The remaining data set is re-averaged and standard deviation is recalculated. All data points 3σ above the new mean are collected. This process repeats until there are data points 3σ above the final mean. The collected data represents particle pulse signals. The remaining data represents background [21,24].
Journal Pre-proof 2.4. Immunoassay procedure AuNPs and AgNPs were prepared according to the literature [25,26]. The detailed synthesis method of AuNPs and AgNPs were shown in the Supplemental Material. The nanoparticle sizes were characterized in Section 3.1. A 500 μL of 600 mg L-1 colloidal gold solution was adjusted to pH 8.2 by adding 0.1 mol L-1 K2 CO 3 and then mixing in 22 μ g of secondary CYFRA21-1 antibody (Ab2-CYFRA21-1). After overnight incubation at 4 ℃, Ab2-CYFRA21-1-AuNPs conjugates were
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stabilized in 1% BSA and incubated for 2 h at 4 ℃. That mixture was centrifuged at 12000 rpm for 20 min. The supernatant was discarded and the sediment re-dispersed in the PBS solution. The
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process was repeated thrice to remove extra antibodies. The Ab2-CYFRA21-1-AuNPs conjugates
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were resuspended in a PBS solution and stored at 4 ℃ for future use. The minimum tagged amount was determined using a flocculation test [27]. Differing Ab2-CYFRA21-1 volumes were added to
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100 μL pH 8.2 AuNPs. After 2 h incubation at room temperature, a 10 μL 10% NaCl solution was
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added and incubated for 1 h. The Ab2-CYFRA21-1, with an additional 10% more than the minimum tagged amount, was added to the 100 μL AuNPs solution and conjugated. The magnetic
Scheme 1
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immunoassay procedure for CYFRA21-1 appears in Scheme 1.
Ab2-CA15-3-AgNPs conjugates were added via a similar method. A 500 μL of 50 mg L-1 silver
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solution was adjusted to pH 8.2 by adding 0.1 mol L-1 K2 CO 3 and mixed with 19.3 μg of secondary CA15-3 antibodies (Ab2-CA15-3). After overnight incubation at 4 ℃, Ab2-CA15-3-AgNPs conjugates were stabilized in 1% BSA and incubated for 2 h at 4 ℃. The mixture was centrifuged at 16000 rpm for 20 min. The supernatant was discarded and the sediment was re-dispersed in a PBS solution. The process was repeated thrice to remove extra antibodies and then the Ab2-CA15-3-AgNPs conjugates were resuspended in a PBS solution and stored at 4 ℃ for further use. A minimum tagged amount was determined using a flocculation test [28]. Ab2-CA15-3, with an additional 10% over the minimum, tagged amount, was added to the 100 μL AgNPs solution for conjugation. The magnetic immunoassay procedure for CA15-3 appears in Scheme 1.
Journal Pre-proof ZnSe QDs were synthesized as published in our previous article [29]. The conjugation of secondary CEA antibody-ZnSe QDs were performed in our previous article [29]. In brief, five hundred microliters of ZnSe QDs were dispersed in 5 mL PBS. Twenty-five mg of EDC and an NHS mixture (1:1) was added to couple ZnSe QDs with the goat anti- mouse IgG antibody (secondary CEA antibody). After reacting for 1 h, the activated ZnSe QDs were collected by centrifugation and washed thrice in a PBS solution. Twenty microliters of Ab2-CEA were spiked and stirred for 2 h. After the reaction, the secondary CEA antibody-ZnSe QDs conjugates
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(Ab2-CEA-ZnSe QDs) were separated by centrifugation and washed thrice with the PBS solution.
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The Ab2-CEA-ZnSe QDs were then blocked with 1% BSA for 1 h a nd stored at 4 ℃. The magnetic
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immunoassay procedure for CEA appears in Scheme 1.
The magnetic immunoassay procedure for CYFRA21-1/CA15-3/CEA appears in Scheme 1.
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Primary CA15-3 antibodies (Ab1-CA15-3), and CEA (Ab1-CEA) along with biotin were mixed at a
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volume ratio of 1:10 and rotated at 15 rpm at room temperature for 4 h [28]. Biotin-Ab1-CEA and biotin-Ab1-CA15-3 were centrifuged and washed thrice with the PBS solution. Seven hundred
μL of 20
U
mL-1
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microliters of 1.5 mg L-1 biotin-Ab1-CYFRA21-1, 500 μL of 10 mg L-1 biotin-Ab1-CEA, and 500 biotin-Ab1-CA15-3
were added
to
1000
μL of 260
mg
L-1
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streptavidin- functionalized magnetic beads (MBs), gently shaken, and incubated for 2 h at room
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temperature. The coated magnetic beads were washed thrice with the PBS solutions to remove unbound biotinylated antibodies and were resuspended in a 1.5 mL PBS solution. A 1% bovine serum albumin (BSA) was added and reacted for 1 h to inhibition non-specific adsorption. The MBs-Ab1 was washed thrice with PBS solution. During the immune reaction process, 10 μL of 50 ng mL-1 CYFRA21-1, 10 μL of 250 mU mL-1 CA15-3, 10 μL of 100 ng mL-1 CEA and 60 μL of 480 mg L-1 MBs-Ab1 were mixed together in one tube. In a second tube, 20 μL of serum samples and 60 μL of MBs-Ab1 were mixed together. The mixture was continuously shaken at 37 ℃ for 1 h to capture target antigens. MBs-Ab1-CYFRA21-1/CEA/CA15-3 was separated using an external magnetic force and washed thrice with the PBS solution. Then, 40 μL of Ab2-CYFRA21-1-AuNPs， 60 μL of Ab2-CEA-ZnSe QDs ， and 60 μL of Ab2-CA15-3-AgNPs were added to the
Journal Pre-proof MBs-Ab1-CYFRA21-1/CEA/CA15-3 followed by 1 h to complete the immune reaction. The MBs-Ab1-CYFRA21-1/CEA/CA15-3-Ab2-AuNPs/ZnSe
QDs/AgNPs
immunocomplex
was
isolated using an external magnetic force and washed thrice with the PBS solution. One hundred microliters
of
1
mol
L-1
formic
acid
were
added
to
the
MBs-Ab1-CYFRA21-1/CEA/CA15-3-Ab2-AuNPs/ZnSe QDs/AgNPs immunocomplex to quantify and then sonicated for 10 min to obtain free AuNPs/ ZnSe QDs/AgNPs [16,29]. The dissociated MBs-Ab1-CYFRA21-1/CEA/CA15-3-Ab2 solutions were separated using a permanent magnet and
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single particle AuNPs/ZnSe QDs/AgNPs were directly introduced into the ICP-MS with a custom,
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gas pressure-assisted, sample introduction system.
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3. Results and discussion
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3.1. Characterization of AuNPs and AgNPs
The synthesized AuNPs and AgNPs were characterized by absorbance spectra, XRD, and TEM.
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AuNPs showed maximal absorbance at 532 nm (Fig. 1a). AgNPs showed maximal absorbance at 418 nm (Fig. 1b). TEM showed AuNPs diameters of about 29 nm (Fig. S1a). AgNPs were about 14
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nm (Fig. S1b). AuNPs diameters were in the 18.3-41.7 nm range with an average diameter of approximately 29 nm which is consistent with TEM results for AuNPs (Fig. 1c). AgNPs with
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diameters in the 6.0-24.0 nm range and an average diameter of approximately 14 nm were formed which is consistent with TEM results for AgNPs (Fig. 1d). An XRD technique characterized AuNPs and AgNPs structures. AuNPs had four peaks at 2θ in the regions of 38.2°, 44.4°, 64.6°, and 77.5° with corresponding indices of (111), (200), (220) and (311) (Fig. S1c). The peaks match that of JCPDS No.04-0784. Five AgNPs characteristic peaks appeared at 2θ in the regions of 38.17°, 44.29°, 64.57°, 77.49°, and 81.65° with corresponding indices of (111), (200), (220), (311) and (222). The peaks match the pattern for JCPDS No.04-0783 (Fig. S1d). Figure 1 AuNPs and AgNPs absorption spectra before, and after, antibody binding appear in Fig. 1a and
Journal Pre-proof 1b, respectively. There was a constant peak pattern after AuNPs bound with Ab2-CYFRA21-1 (Fig. 1a). A red shift of 8 nm for the maximum absorption of AuNPs occurred indicating Ab2-CYFRA21-1-AuNPs conjugates were obtained. The maximum absorption for AgNPs was at 418 nm and shifted to 439 nm after binding with Ab2-CA15-3 (Fig. 1b). A flocculation test optimized the amount of antibodies added to AuNPs or AgNPs for conjugation. The 10% NaCl solution caused AuNPs flocculation and decreased UV absorption by AuNPs, if they were unsaturated.
The
stability
of
Ab2-CYFRA21-1-labeled
AuNPs
was
improved
once
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Ab2-CYFRA21-1 levels were sufficiently high. No obvious AuNPs UV absorption change was
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observed.
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Figure 2
AuNPs absorption increased as the amount of Ab2-CYFRA21-1 increased until reaching 4 μL
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(Fig. 2a). This corresponds to an absolute amount of 4 μg. Finally, 4.4 μg Ab2-CYFRA21-1 was
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added to 100 μL of 600 mg L-1 AuNPs for conjugation. Similar results were obtained for AgNPs, and 3.85 μg Ab2-CA15-3 was added to 100 μL of 50 mg L-1 AgNPs for conjugation (Fig. 2b).
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3.2. Selection of SP-ICP-MS conditions
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SP-ICP-MS analysis requires that a single particle at a time enter plasma, so that each mass spectrum transient signal corresponds to a single particle. Dwell time duration was chosen to meet
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this target. The number of AuNPs detection events per second decreased as dwell time increased from 0.1 ms to 10 ms (Fig. 3a). Mean particle intensity increased as dwell time duration increased due to signal overlap of the multiple nanoparticles (Fig. 3a). A dwell time of 0.1 ms was chosen for AuNPs. Similar results obtained for AgNPs when a dwell time of 0.1 ms was chosen for AgNPs (Fig. 3b). Optimized ZnSe QD dwell times were discussed in our previous article [29]. A dwell time of 0.1 ms was chosen. Figure 3 The effects of AuNPs concentrations on the number of detection events appear in Fig. S2a. The number of detection events increased with as AuNPs concentrations increased. They remained
Journal Pre-proof constant when AuNPs concentrations were higher than 600 ng L-1 . Mean particle intensity increased as AuNPs concentrations increased due to agglomeration. The maximum AuNPs concentration was 600 ng L-1 . Similar results were obtained for AgNPs when the maximum AgNPs concentration was 1000 ng L-1 (Fig. S2b). 3.3. Spectral interference The most abundant zinc (64 Zn) suffered from severe interference from 64 Ni, 32 S16 O16O, 48 Ca16 O, S S, 31 P16 O17O, and 36 Ar14N 14N [30]. Methane (CH4 ) was used here as the reaction gas to alleviate
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32 32
spectral interference [31]. CH4 was selected as the reaction gas with a CH4 flow rate of 1.0 mL min-1 197
Au and
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[29]. Au and Ag were detected using the isotopes
107
Ag at abundances of 100% and
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51.35%. A high level of interference may be produced from 181 Ta16 O and 91 Zr16 O to 197 Au and 107 Ag. Ta and Zr in human lung cancer serum samples and PBS solutions have not been found. 91
Zr16 O effects overlap with
197
107
Ta16 O
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and
181
Au and
Ag signals were negligible.
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3.4. Optimization of immunoassay conditions
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Several immunoassay procedure parameters were examined, including: BSA volume; MBs-Ab1-CYFRA21-1/CEA/CA15-3, amounts;
and,
Ab2-CEA-ZnSe
QD
and
MBs-Ab1-CYFRA21-1/CEA/CA15-3,
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Ab2-CA15-3-AgNPs,
Ab2-CYFRA21-1-AuNPs,
Ab2-CYFRA21-1-AuNPs, Ab2-CEA-ZnSe QDs, and Ab2-CA15-3-AgNPs incubation times to
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obtain optimal analytical performances. A 1% (m/v) BSA solution was used as a blocking reagent to eliminate nonspecific adsorption. BSA blocking volume effects on Au/Ag/Zn intensity was examined using the immunoassay procedure described above, except for the CYFRA21-1/CEA/CA15-3 incubation step. When BSA amounts insufficiently blocked nonspecific adsorption, Ab2-CYFRA21-1-AuNPs, Ab2-CEA-ZnSe QDs, and Ab2-CA15-3-AgNPs residue concentrations increased as BSA volume increased. Au/Zn/Ag intensity increased as BSA volume increased. Blocking was complete when volume reached 600 μL (Fig. 4). Figure 4
Journal Pre-proof The volume and incubation times of MBs-Ab1-CYFRA21-1/CEA/CA15-3 were optimized to obtain high capture efficiency. This was achieved by fixing MBs-Ab1-CYFRA21-1/CEA/CA15-3 concentrations at 480 mg L-1 , and CYFRA21-1, CEA and CA15-3 concentrations at 50 ng mL-1 , 100 ng mL-1 and 250 mU mL-1 , respectively. CYFRA21-1, CEA and CA15-3 standard solutions with 10 μL was used. Ab2-CYFRA21-1-AuNPs, Ab2-CEA- ZnSe QDs, and Ab2-CA15-3-AgNPs volumes were 40 μL, 60 μL, and 60 μL, respectively. Incubation time was 1 h. The effect of MBs-Ab1-CYFRA21-1/CEA/CA15-3 volumes ranging from 20 μL to 80 μL on immunoreaction
197
Au/64 Zn/107 Ag increased rapidly with the increase of volume
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The number of detection events for
of
between MBs-Ab1-CYFRA21-1/CEA/CA15-3 and CYFRA21-1/CEA/CA15-3 was investigated.
for MBs-Ab1-CYFRA21-1/CEA/CA15-3 from 20 μL to 60 μL and then decreased from 60 μL to 80
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μL (Fig. 5a). A volume of 60 μL of MBs-Ab1-CYFRA21-1/CEA/CA15-3 was used to capture
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CYFRA21-1/CEA/CA15-3. Incubation time duration effects on immunoreactions between
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MBs-Ab1-CYFRA21-1/CEA/CA15-3 and CYFRA21-1/CEA/CA15-3 in the 20-120 min range was investigated. The results appear in Fig. 5b. The number of
197
Au/64 Zn/107 Ag detection events
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increased from 20 min to 60 min and remained constant after incubation for 60 min. This indicates immunoreaction completed in by the 60 min mark. An incubation time of 60 min was used in the
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following experiments.
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Figure 5
It was essential to add additional Ab2-CYFRA21-1-AuNPs, Ab2-CEA-ZnSe QDs, and Ab2-CA15-3-AgNPs to the reaction system. This was done by fixing Ab2-CYFRA21-1-AuNPs, Ab2-CEA-ZnSe QDs and Ab2-CA15-3-AgNPs concentrations at 22, 20 and 20 μg mL-1 , respectively.
The
effects
of
Ab2-CYFRA21-1-AuNPs,
Ab2-CEA-ZnSe
QDs
Ab2-CA15-3-AgNPs volumes were investigated for the 10 to 80 μL range. The number of
and 197
Au
detection events increased rapidly as Ab2-CYFRA21-1-AuNPs volume increased from 10 μL to 40 μL. It remained constant from 40 μL to 80 μL (Fig 6a). Forty μL of Ab2-CYFRA21-1-AuNPs was used. Similar results were obtained for Ab2-CEA-ZnSe QDs and Ab2-CA15-3-AgNPs. A volume of 60 μL of Ab2-CEA-ZnSe QDs and Ab2-CA15-3-AgNPs was used in the further experiments (Fig.
Journal Pre-proof 6a). The number of 197 Au/64 Zn/107 Ag detection events obtained at different incubation times ranging from 20 to 120 min appears in Fig. 6b.
197
Au/64 Zn/107 Ag detection events increased between 20 min
and 60 min, but remained constant after incubation for 60 min. This ind icates that the immunoreaction is completed within 60 min. A 60 min incubation time was used. Figure 6 3.5. Performance of analytical method
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Under the optimized experimental conditions, the analytical performance of the method was
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evaluated for simultaneous quantification of the three biomarkers. The plot between the number of detection events and CYFRA21-1/CEA/CA15-3 concentration appears in Fig. 7. The dynamic
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ranges were 0.05-50 ng mL-1 for CYFRA21-1, 0.02-100 ng mL-1 for CEA, 0.6-250 mU mL-1 for
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CA15-3. Linear equations correlation coefficients were 0.9999, 0.9987, and 0.9970 for CYFRA21-1, CEA, and CA15-3, respectively. Our method’s detection limits were 0.02 ng mL-1 , 0.006 ng mL-1
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and 0.25 mU mL-1 for CYFRA21-1, CEA and CA15-3, respectively. The relative standard deviation (RSD) for the six replicate determinations was 3.2%, 4.4%, and 4.2% for CYFRA21-1, CEA and
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CA15-3, respectively. A comparison of the immunoassay methods for CYFRA21-1, CEA, and
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CA15-3 determination appear in Table S1. The detection limit of the present method for CYFRA21-1 is lower than previous immunoassays [7,8,32,33] and higher than that reported by
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Wang et al. [22]. The present method’s detection limit for CEA was lower than previous reports [7,8,22,32]. The detection limit of the present method for CA15-3 was lower than previous results [6,13,34,35]. Figure 7 3.6. Sample analysis The feasibility of this method was investigated. The concentrations of CYFRA21-1, CEA, and CA15-3 in human lung cancer serum samples (from the Guilin Fifth People’s Hospital (Guilin, China)) were tested by this method. For determination, serum samples spiked with standard CYFRA21-1, CEA, and CA15-3 were analyzed using a single particle mode ICP-MS. The results
Journal Pre-proof appear in Table 2. The recovery for CYFRA21-1, CEA and CA15-3 ranged from 89.8%-101%. Table 2
4. Conclusions In this work, we first reported an ICP-MS-based multiple immunoassay using AuNPs, AgNPs, and ZnSe QDs as element tags for simultaneous determination of CYFRA21-1, CA15-3, and CEA
of
in human lung cancer serum samples. Highly efficient captures of CYFRA21-1, CA15-3, and CEA
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were obtained using streptavidin- functionalized magnetic beads conjugated with a primary antibody.
-p
AuNPs, AgNPs, and ZnSe QDs-tagged second antibodies were used to determine the target antigen using SP-ICP-MS. The method was successfully applied to detect CYFRA21-1, CA15-3, and CEA
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in human lung cancer serum samples. This method is expected to have applications to other
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biomarker detection. Comparing to one element (Ag, Au, Zn), the simultaneous immunoassay of
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Acknowledgements
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multianalytes can improve assay efficiencies and reduce sample consumption.
This work was financially supported by the National Natural Science Foundation of China
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(21765004) and by the Guangxi Science Foundation of China (2019GXNSFAA245076), and by Guangxi University of Chinese Medicine Research Foundation (2018BS017). The research fund of State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources (Guangxi Normal University) (CMEMR2018-C18) is gratefully acknowledged.
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carbon quantum dots and signal amplification, Biosens. Bioelectron. 89 (2017) 453-460. [26] C.-H. Yeh, W.-T. Chen, H.-P. Lin, T.-C. Chang, Y.-C. Lin, Development of an immunoassay
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based on impedance measurements utilizing an antibody-nanosilver probe, silver enhancement, and electro-microchip, Sensor. Actuat. B-Chem. 139 (2009) 387-393. [27] C. Zhang, Z. Zhang, B. Yu, J. Shi, X. Zhang, Application of the biological conjugate between antibody and colloid Au nanoparticles as analyte to inductively coupled plasma mass spectrometry, Anal. Chem. 74 (2002) 96-99. [28] S. Shan, Z. Zhong, W. Lai, Y. Xiong, X. Cui, D. Liu, Immunomagnetic nanobeads based on a streptavidin-biotin system for the highly efficient and specific separation of Listeria monocytogenes, Food Control 45 (2014) 138-142. [29] Y. Cao, G. Mo, J. Feng, X. He, L. Tang, C. Yu, B. Deng, Based on ZnSe q uantum dots labeling
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human serum using a sandwich-type ICP-MS immunoassay, Bull. Korean Chem. Soc. 37 (2016)
Journal Pre-proof List of Tables and Figures Table 1. ICP-MS instrument operating conditions. Table 2. Analytical results of human lung cancer serum samples (n=3). Scheme 1. Sandwich-type magnetic immunoassay using antibody-immobilized magnetic nanoparticles. Figure 1. Characterization of AuNPs and AgNPs. AuNPs and Ab2-CYFRA21-1-AuNPs conjugates UV-Vis spectrum (a). AgNPs and Ab2-CA15-3-AgNPs conjugates UV-Vis spectrum (b). AuNPs (c) and AgNPs (d) size
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distributions.
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Figure 2. Effects of Ab2-CYFRA21-1 amount (a) and Ab2-CA15-3 amount (b) on absorbance.
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Figure 3. Number of detection events per second and mean particle intens ity as a function of dwell time for
Figure
4.
BSA
blocking
volume
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AuNPs (a) and AgNPs (b) dwell times.
effects.
MBs-Ab1-CYFRA21-1/CEA/CA15-3:
480
mg
L-1 ,
-1
-1
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Ab2-CYFRA21-1-AuNPs, Ab2-CEA-ZnSe QDs and Ab2-CA15-3-AgNPs concentrations at 22 μg mL , 20 μg -1
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mL and 20 μg mL , respectively. Incubation time was 1 h. Error bar represents s.d. value for triplicate analysis. Figure 5. MBs-Ab1 volume (a) and incubation time (b) effects on number of detection events. CYFRA21-1, CEA
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and CA15-3 standard solutions with 10 μL was used. Ab2-CEA-ZnSe QDs and Ab2-CA15-3-AgNPs
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concentrations at 22, 20 and 20 μg mL-1 , respectively. Error bar represents s.d. value for triplicate analysis. Figure 6. Labeled-Ab2 volume effects on the number of detection events (a) and incubation time effects on the number of detection events (b). MBs-Ab1-CYFRA21-1/CEA/CA15-3: 480 mg L-1 , CYFRA21-1, CEA and CA15-3 standard solutions with 10 μL was used. Error bar represents s.d. value for triplicate analysis. Figure 7. CYFRA21-1, CA15-3 and CEA calibration plot using SP-ICP-MS.
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Table 1. ICP-MS instrument operating conditions. Value
RF power (W)
1000
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Parameter
Nebulizer gas flow rate (L min-1 )
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Auxiliary gas flow rate (L min-1 )
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Plasma gas flow rate (L min-1 )
Scan mode
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RPq
0.9 1.20 15
Au, Ag: 0.5; Zn: 0.8 Peak hopping
Sample uptake rate (μL min-1 )
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7.5 0.1
Sweeps
1
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Dwell time (ms)
Cell gas flow rate DRC-CH4 (mL min-1 )
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Monitored isotope (m/z)
1.0 197
Au,
107
Ag,
64
Zn
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(ng mL-1 )
(%)
(%)
16.9±0.08
91.8
3.5
14.6±0.10
89.8
4.3
10.0
9.88±0.05
98.8
3.8
15.0
27.0±0.05
95.3
4.2
6.73±0.08
10.0
16.2±0.03
94.7
4.0
---
10.0
10.1±0.12
101
3.1
18.3±0.05
20.0
37.3±0.09
95.0
3.8
CEA
8.11±0.05
10.0
18.1±0.05
99.9
3.8
CA15-3
1.19±0.03
2.00
2.99±0.02
90.0
3.5
Serum 2
(ng mL-1 )
7.72±0.05
10.0
CEA
5.62±0.15
CA15-3
---
CYFRA21-1
(male, 65 yrs)
12.7±0.07
CEA CA15-3 CYFRA21-1
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Serum 3
(ng mL-1 )
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(female, 66 yrs)
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Note: mU mL-1 for CA15-3
Total found
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(male, 56 yrs)
Added
10.0
-p
CYFRA21-1
Original
of
RSD
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Sample
Serum 1
Recovery
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Table 2. Analytical results of human lung cancer serum samples (n=3).
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Author Contribution Statement Yupin Cao: Conceptualization, Methodology, Investigation, Writing - Original Draft. Jinsu Feng: Visualization, Writing - Review and Editing. Lifu Tang: Data analysis, Writing – review & editing.
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Guichun Mo: Writing - Review and Editing. Weiming Mo: Visualization. Biyang Deng:
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Supervision.
Journal Pre-proof Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
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Highlights · The AuNPs, ZnSeQDs and AgNPs as elemental tags in sandwich-type immunoassay
· SP-ICP-MS was used to detect AuNPs, ZnSe QDs and AgNPs at the immunocomplexes
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· The proposed method succeeded simultaneous detection of CYFRA21-1, CEA and CA15-3
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· Total volume of immunocomplex solution was 10 μL for analysis.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Yupin Cao, Jinsu Feng, Lifu Tang, Guichun Mo, Weiming Mo, Biyang Deng PII:
S0584-8547(19)30479-3
DOI:
https://doi.org/10.1016/j.sab.2020.105797
Reference:
SAB 105797
To appear in:
Spectrochimica Acta Part B: Atomic Spectroscopy
Received date:
10 October 2019
Revised date:
15 February 2020
Accepted date:
17 February 2020
Please cite this article as: Y. Cao, J. Feng, L. Tang, et al., Detection of three tumor biomarkers in human lung cancer serum using single particle ICP-MS combined with magnetic immunoassay, Spectrochimica Acta Part B: Atomic Spectroscopy(2019), https://doi.org/10.1016/j.sab.2020.105797
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© 2019 Published by Elsevier.
Journal Pre-proof
Detection of three tumor biomarkers in human lung cancer serum using single particle ICP-MS combined with magnetic immunoassay Yupin Cao1,2 , Jinsu Feng1 , Lifu Tang1 , Guichun Mo1 , Weiming Mo1 , Biyang Deng1,* [email protected] 1
State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources,
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School of Chemistry and Pharmaceutical Sciences, Guangxi Normal University, Guilin 541004, China
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Guangxi University of Chinese Medicine, Nanning 530200, China
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2
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* Corresponding Author.
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Telephone: +86-773-5845726, Fax: +86-773-2120958,
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Email: [email protected] (B. Deng)
Journal Pre-proof
Abstract A new method
for simultaneously determining cytokeratin fragment antigen 21-1
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(CYFRA21-1), carcinoembryonic antigen (CEA), and carbohydrate antigen (CA15-3) was
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developed using single-particle, ICP-MS-based, magnetic immunoassay. Gold nanoparticles (AuNPs), ZnSe quantum dots (QDs), and Silver nanoparticles (AgNPs) were employed to tag
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antibodies. A highly efficient capture of CYFRA21-1, CEA, and CA15-3 was achieved using
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biotinylated, antibody-coated magnetic beads. AuNPs/ZnSe QDs/AgNPs-tagged second antibodies helped realize sensitive quantification of the target antigen using single particle ICP-MS. Under
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optimum conditions, CYFRA21-1, CEA, and CA15-3 linear concentration ranges were 0.05-50 ng mL-1 , 0.02-100 ng mL-1 , and 0.6-250 mU mL-1 . The correlation coefficients were 0.9999, 0.9987,
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and 0.9970, respectively. CYFRA21-1, CEA, and CA15-3 detection limits were 0.02 ng mL-1 , 0.006
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ng mL-1 , and 0.25 mU mL-1 , respectively. Relative standard deviations (RSD) for CYFRA21-1, CEA, and CA15-3 were 3.2%, 4.4%, and 4.2%, respectively. The method simultaneously detected
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CYFRA21-1, CEA, and CA15-3 in human lung cancer serum samples with recoveries of between 89.8% and 101%.
Keywords: CYFRA21-1; CEA; CA15-3; Single particle-ICP-MS; Magnetic immunoassay
Journal Pre-proof 1. Introduction Cancer is commonly fatal to humans [1]. Lung cancers continue as the most common, lethal cancer and account for more than 25% of all U.S. cancer deaths [2]. It is classified into two major groups: small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) [3]. Presently cytokeratin fragment antigen 21-1 (CYFRA21-1) is the preferred biomarker for detecting NSCLC, particularly squamous cell carcinoma [4]. Carcinoembryonic antigen (CEA) is an accepted
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tumor marker for colorectal cancer. It has been reported as a prognostic lung cancer marker [5]. Carbohydrate antigen (CA15-3), encoded by MUC1, which is known as a tumor marker for breast
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cancer [6]. Tumor biomarkers mirror tumor tissue, cell differentiation, and cell function. Early stage
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detection of tumor biomarkers contributes to diagnosis, classification, prognosis judgment, and
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treatment [7].
Several methods have been used to analyze biomarkers, including: enzyme-linked
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immunosorbent assay (ELISA) [8]; fluoroimmunoassay (FIA) [9]; chemiluminescent immunoassay (CLIA) [10,11]; electrochemical immunoassay (ECIA) [12,13]; and inductively coupled plasma
na
mass spectrometry (ICP-MS) [14-19]. An element-specific detector, ICP-MS is now one of the most powerful elemental analysis tools having the advantages of high sensitivity, low matrix effects, wide
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dynamic range, and multiplex detection ability [20,21]. Since the time resolved transient signals
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induced by the flash of ions arising from the ionization in the plasma torch can reflect the concentrations of nanoparticles, the concentration of nanoparticle-tagged immunocomplex could be quantified by the frequency of transient signals [18]. ICP-MS element tagging has the potential to achieve multiplex analysis for massive bioassays and clinical diagnosis [18]. Detecting one biomarker at a time is very time-consuming when measuring multiple biomarkers. The simultaneous immunoassay of multianalytes has attracted much attention. It improves assay efficiencies and lowers sample consumption [22]. Hong et al. reported on an ICP-MS-based multiple magnetic immunoassay which simultaneously detected three gynecological tumor biomarkers in clinical serum samples. A highly efficient capture of HER-2, HE4, and CA15-3 using biotinylated antibody-coated magnetic beads was obtained. Using a lanthanide ion-tagged, second antibody
Journal Pre-proof helped realize a sensitive quantification of the target antigen using ICP-MS [6]. Ko et al. reported a multiplex detection of biomarkers via a sandwich-type immunoassay for AFP, NSE, and CRP [15]. Zhang et al. reported an ICP-MS-based immunoassay using golden nanoparticles (AuNPs) and silver nanoparticles (AgNPs) to tag elements for determining CEA and AFP in human serum samples [16]. This article first reports on an ICP-MS-based, multiple immunoassay using AuNPs, AgNPs, and
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ZnSe quantum dots (QDs) to tag elements for simultaneously determining CYFRA21-1, CA15-3, and CEA in human lung cancer serum samples. First, streptavidin- functionalized magnetic beads
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were conjugated with a primary antibody to capture target proteins. Second, analyte quantification
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was realized via a sandwich-type immunoreaction using streptavidin- functionalized magnetic beads and a biotinylated antibody. AuNPs, AgNPs, and ZnSe QDs were synthesized as probes to determine
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CYFRA21-1, CA15-3, and CEA in human lung cancer serum samples via SP-ICP-MS. The
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suspension solutions were separated by a permanent magnet. Single particles were direc tly introduced into ICP-MS with a custom, gas pressure-assisted, sample introduction system which was
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successfully used on human lung cancer serum samples. Comparing to tag one element, the
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2. Experimental
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immunoassay of multi-elements can improve assay efficiency and reduce sample consumption.
2.1. Instrumentation
A NexIon 300X ICP-MS system (Perkin Elmer, USA) with a custom, gas pressure-assisted sample- introduction system (GPASIS) [23] determined
197
Au,
107
Ag, and
64
Zn in a single particle
mode. The optimized ICP-MS operating parameters appear in Table 1. AuNPs, AgNPs and ZnSe QDs were characterized using Fourier transform infrared spectroscopy (FTIR, Perkin Elmer, USA), transmission electron microscope (TEM, JEM-2100, JEOL, Japan), and a Rigaku D/max 2500/pc X-ray powder diffractometer (XRD, Rigaku, Japan). Table 1
Journal Pre-proof 2.2. Reagents N-hydroxysuccinimide
(NHS,
98%),
N-(3-(dimethylamino)propyl)-N’-ethylcarbodiimide
hydrochloride (EDC, 98%) was obtained from Aladdin Industrial Corporation (Shanghai, China). Bovine serum albumin (BSA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Streptavidin functionalized magnetic beads, biotinylated monoclonal CYFRA21-1 antibody (goat, Ab1-CYFRA21-1), and CYFRA21-1 were from Guangxi Jia Ye Technology Co., Ltd (Guangxi,
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China). Rabbit anti-goat IgG antibody (Ab2-CYFRA21-1), CEA, mouse anti-CEA antibody (Ab1-CEA) and goat anti- mouse IgG antibody (Ab2-CEA), CA15-3, rabbit anti-MUC1
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(Ab1-CA15-3) and goat anti-rabbit IgG (Ab2-CA15-3) were purchased from Bioss (Beijing, China).
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Methane (99.999%) was from Dalian Special Gases (Liaoning, China). Formic acid, sodium borohydride (NaBH4 ), zinc chloride (ZnCl2 ), sodium chloride (NaCl), potassium chloride (KCl),
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di-potassium hydrogen phosphate trihydrate (K 2 HPO 4 ∙3H2 O), potassium dihydrogen phosphate
lP
(KH2 PO4 ) were supplied by Xilong Chemical of Guangdong (Guangdong, China). ICP-MS daily performance was checked using a multielement standard solution containing Be, Mg, In, U and Ce
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at concentrations of 1 ng mL-1 (Perkin Elmer, USA). Ultrapure water (18.2 MΩ) was used. All solutions were filtered through a 0.45 μm membrane filter before use, which supplied by Shanghai
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Xinya Purification Material Factory (Shanghai, China).
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2.3. SP-ICP-MS data processing An iterative algorithm, based on a three-time standard deviation (3σ), was used to discriminate particle pulse from the background signal for SP-ICP-MS. The determining particle events first step begins by averaging the entire data set and collecting all data points that are 3σ above the mean for the entire set. This is similar to outlier removal from a normally-distributed data set. Near-normal distribution data point distribution is assumed. The remaining data set is re-averaged and standard deviation is recalculated. All data points 3σ above the new mean are collected. This process repeats until there are data points 3σ above the final mean. The collected data represents particle pulse signals. The remaining data represents background [21,24].
Journal Pre-proof 2.4. Immunoassay procedure AuNPs and AgNPs were prepared according to the literature [25,26]. The detailed synthesis method of AuNPs and AgNPs were shown in the Supplemental Material. The nanoparticle sizes were characterized in Section 3.1. A 500 μL of 600 mg L-1 colloidal gold solution was adjusted to pH 8.2 by adding 0.1 mol L-1 K2 CO 3 and then mixing in 22 μ g of secondary CYFRA21-1 antibody (Ab2-CYFRA21-1). After overnight incubation at 4 ℃, Ab2-CYFRA21-1-AuNPs conjugates were
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stabilized in 1% BSA and incubated for 2 h at 4 ℃. That mixture was centrifuged at 12000 rpm for 20 min. The supernatant was discarded and the sediment re-dispersed in the PBS solution. The
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process was repeated thrice to remove extra antibodies. The Ab2-CYFRA21-1-AuNPs conjugates
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were resuspended in a PBS solution and stored at 4 ℃ for future use. The minimum tagged amount was determined using a flocculation test [27]. Differing Ab2-CYFRA21-1 volumes were added to
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100 μL pH 8.2 AuNPs. After 2 h incubation at room temperature, a 10 μL 10% NaCl solution was
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added and incubated for 1 h. The Ab2-CYFRA21-1, with an additional 10% more than the minimum tagged amount, was added to the 100 μL AuNPs solution and conjugated. The magnetic
Scheme 1
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immunoassay procedure for CYFRA21-1 appears in Scheme 1.
Ab2-CA15-3-AgNPs conjugates were added via a similar method. A 500 μL of 50 mg L-1 silver
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solution was adjusted to pH 8.2 by adding 0.1 mol L-1 K2 CO 3 and mixed with 19.3 μg of secondary CA15-3 antibodies (Ab2-CA15-3). After overnight incubation at 4 ℃, Ab2-CA15-3-AgNPs conjugates were stabilized in 1% BSA and incubated for 2 h at 4 ℃. The mixture was centrifuged at 16000 rpm for 20 min. The supernatant was discarded and the sediment was re-dispersed in a PBS solution. The process was repeated thrice to remove extra antibodies and then the Ab2-CA15-3-AgNPs conjugates were resuspended in a PBS solution and stored at 4 ℃ for further use. A minimum tagged amount was determined using a flocculation test [28]. Ab2-CA15-3, with an additional 10% over the minimum, tagged amount, was added to the 100 μL AgNPs solution for conjugation. The magnetic immunoassay procedure for CA15-3 appears in Scheme 1.
Journal Pre-proof ZnSe QDs were synthesized as published in our previous article [29]. The conjugation of secondary CEA antibody-ZnSe QDs were performed in our previous article [29]. In brief, five hundred microliters of ZnSe QDs were dispersed in 5 mL PBS. Twenty-five mg of EDC and an NHS mixture (1:1) was added to couple ZnSe QDs with the goat anti- mouse IgG antibody (secondary CEA antibody). After reacting for 1 h, the activated ZnSe QDs were collected by centrifugation and washed thrice in a PBS solution. Twenty microliters of Ab2-CEA were spiked and stirred for 2 h. After the reaction, the secondary CEA antibody-ZnSe QDs conjugates
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(Ab2-CEA-ZnSe QDs) were separated by centrifugation and washed thrice with the PBS solution.
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The Ab2-CEA-ZnSe QDs were then blocked with 1% BSA for 1 h a nd stored at 4 ℃. The magnetic
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immunoassay procedure for CEA appears in Scheme 1.
The magnetic immunoassay procedure for CYFRA21-1/CA15-3/CEA appears in Scheme 1.
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Primary CA15-3 antibodies (Ab1-CA15-3), and CEA (Ab1-CEA) along with biotin were mixed at a
lP
volume ratio of 1:10 and rotated at 15 rpm at room temperature for 4 h [28]. Biotin-Ab1-CEA and biotin-Ab1-CA15-3 were centrifuged and washed thrice with the PBS solution. Seven hundred
μL of 20
U
mL-1
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microliters of 1.5 mg L-1 biotin-Ab1-CYFRA21-1, 500 μL of 10 mg L-1 biotin-Ab1-CEA, and 500 biotin-Ab1-CA15-3
were added
to
1000
μL of 260
mg
L-1
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streptavidin- functionalized magnetic beads (MBs), gently shaken, and incubated for 2 h at room
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temperature. The coated magnetic beads were washed thrice with the PBS solutions to remove unbound biotinylated antibodies and were resuspended in a 1.5 mL PBS solution. A 1% bovine serum albumin (BSA) was added and reacted for 1 h to inhibition non-specific adsorption. The MBs-Ab1 was washed thrice with PBS solution. During the immune reaction process, 10 μL of 50 ng mL-1 CYFRA21-1, 10 μL of 250 mU mL-1 CA15-3, 10 μL of 100 ng mL-1 CEA and 60 μL of 480 mg L-1 MBs-Ab1 were mixed together in one tube. In a second tube, 20 μL of serum samples and 60 μL of MBs-Ab1 were mixed together. The mixture was continuously shaken at 37 ℃ for 1 h to capture target antigens. MBs-Ab1-CYFRA21-1/CEA/CA15-3 was separated using an external magnetic force and washed thrice with the PBS solution. Then, 40 μL of Ab2-CYFRA21-1-AuNPs， 60 μL of Ab2-CEA-ZnSe QDs ， and 60 μL of Ab2-CA15-3-AgNPs were added to the
Journal Pre-proof MBs-Ab1-CYFRA21-1/CEA/CA15-3 followed by 1 h to complete the immune reaction. The MBs-Ab1-CYFRA21-1/CEA/CA15-3-Ab2-AuNPs/ZnSe
QDs/AgNPs
immunocomplex
was
isolated using an external magnetic force and washed thrice with the PBS solution. One hundred microliters
of
1
mol
L-1
formic
acid
were
added
to
the
MBs-Ab1-CYFRA21-1/CEA/CA15-3-Ab2-AuNPs/ZnSe QDs/AgNPs immunocomplex to quantify and then sonicated for 10 min to obtain free AuNPs/ ZnSe QDs/AgNPs [16,29]. The dissociated MBs-Ab1-CYFRA21-1/CEA/CA15-3-Ab2 solutions were separated using a permanent magnet and
of
single particle AuNPs/ZnSe QDs/AgNPs were directly introduced into the ICP-MS with a custom,
-p
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gas pressure-assisted, sample introduction system.
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3. Results and discussion
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3.1. Characterization of AuNPs and AgNPs
The synthesized AuNPs and AgNPs were characterized by absorbance spectra, XRD, and TEM.
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AuNPs showed maximal absorbance at 532 nm (Fig. 1a). AgNPs showed maximal absorbance at 418 nm (Fig. 1b). TEM showed AuNPs diameters of about 29 nm (Fig. S1a). AgNPs were about 14
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nm (Fig. S1b). AuNPs diameters were in the 18.3-41.7 nm range with an average diameter of approximately 29 nm which is consistent with TEM results for AuNPs (Fig. 1c). AgNPs with
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diameters in the 6.0-24.0 nm range and an average diameter of approximately 14 nm were formed which is consistent with TEM results for AgNPs (Fig. 1d). An XRD technique characterized AuNPs and AgNPs structures. AuNPs had four peaks at 2θ in the regions of 38.2°, 44.4°, 64.6°, and 77.5° with corresponding indices of (111), (200), (220) and (311) (Fig. S1c). The peaks match that of JCPDS No.04-0784. Five AgNPs characteristic peaks appeared at 2θ in the regions of 38.17°, 44.29°, 64.57°, 77.49°, and 81.65° with corresponding indices of (111), (200), (220), (311) and (222). The peaks match the pattern for JCPDS No.04-0783 (Fig. S1d). Figure 1 AuNPs and AgNPs absorption spectra before, and after, antibody binding appear in Fig. 1a and
Journal Pre-proof 1b, respectively. There was a constant peak pattern after AuNPs bound with Ab2-CYFRA21-1 (Fig. 1a). A red shift of 8 nm for the maximum absorption of AuNPs occurred indicating Ab2-CYFRA21-1-AuNPs conjugates were obtained. The maximum absorption for AgNPs was at 418 nm and shifted to 439 nm after binding with Ab2-CA15-3 (Fig. 1b). A flocculation test optimized the amount of antibodies added to AuNPs or AgNPs for conjugation. The 10% NaCl solution caused AuNPs flocculation and decreased UV absorption by AuNPs, if they were unsaturated.
The
stability
of
Ab2-CYFRA21-1-labeled
AuNPs
was
improved
once
of
Ab2-CYFRA21-1 levels were sufficiently high. No obvious AuNPs UV absorption change was
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observed.
-p
Figure 2
AuNPs absorption increased as the amount of Ab2-CYFRA21-1 increased until reaching 4 μL
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(Fig. 2a). This corresponds to an absolute amount of 4 μg. Finally, 4.4 μg Ab2-CYFRA21-1 was
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added to 100 μL of 600 mg L-1 AuNPs for conjugation. Similar results were obtained for AgNPs, and 3.85 μg Ab2-CA15-3 was added to 100 μL of 50 mg L-1 AgNPs for conjugation (Fig. 2b).
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3.2. Selection of SP-ICP-MS conditions
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SP-ICP-MS analysis requires that a single particle at a time enter plasma, so that each mass spectrum transient signal corresponds to a single particle. Dwell time duration was chosen to meet
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this target. The number of AuNPs detection events per second decreased as dwell time increased from 0.1 ms to 10 ms (Fig. 3a). Mean particle intensity increased as dwell time duration increased due to signal overlap of the multiple nanoparticles (Fig. 3a). A dwell time of 0.1 ms was chosen for AuNPs. Similar results obtained for AgNPs when a dwell time of 0.1 ms was chosen for AgNPs (Fig. 3b). Optimized ZnSe QD dwell times were discussed in our previous article [29]. A dwell time of 0.1 ms was chosen. Figure 3 The effects of AuNPs concentrations on the number of detection events appear in Fig. S2a. The number of detection events increased with as AuNPs concentrations increased. They remained
Journal Pre-proof constant when AuNPs concentrations were higher than 600 ng L-1 . Mean particle intensity increased as AuNPs concentrations increased due to agglomeration. The maximum AuNPs concentration was 600 ng L-1 . Similar results were obtained for AgNPs when the maximum AgNPs concentration was 1000 ng L-1 (Fig. S2b). 3.3. Spectral interference The most abundant zinc (64 Zn) suffered from severe interference from 64 Ni, 32 S16 O16O, 48 Ca16 O, S S, 31 P16 O17O, and 36 Ar14N 14N [30]. Methane (CH4 ) was used here as the reaction gas to alleviate
of
32 32
spectral interference [31]. CH4 was selected as the reaction gas with a CH4 flow rate of 1.0 mL min-1 197
Au and
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[29]. Au and Ag were detected using the isotopes
107
Ag at abundances of 100% and
-p
51.35%. A high level of interference may be produced from 181 Ta16 O and 91 Zr16 O to 197 Au and 107 Ag. Ta and Zr in human lung cancer serum samples and PBS solutions have not been found. 91
Zr16 O effects overlap with
197
107
Ta16 O
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and
181
Au and
Ag signals were negligible.
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3.4. Optimization of immunoassay conditions
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Several immunoassay procedure parameters were examined, including: BSA volume; MBs-Ab1-CYFRA21-1/CEA/CA15-3, amounts;
and,
Ab2-CEA-ZnSe
QD
and
MBs-Ab1-CYFRA21-1/CEA/CA15-3,
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Ab2-CA15-3-AgNPs,
Ab2-CYFRA21-1-AuNPs,
Ab2-CYFRA21-1-AuNPs, Ab2-CEA-ZnSe QDs, and Ab2-CA15-3-AgNPs incubation times to
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obtain optimal analytical performances. A 1% (m/v) BSA solution was used as a blocking reagent to eliminate nonspecific adsorption. BSA blocking volume effects on Au/Ag/Zn intensity was examined using the immunoassay procedure described above, except for the CYFRA21-1/CEA/CA15-3 incubation step. When BSA amounts insufficiently blocked nonspecific adsorption, Ab2-CYFRA21-1-AuNPs, Ab2-CEA-ZnSe QDs, and Ab2-CA15-3-AgNPs residue concentrations increased as BSA volume increased. Au/Zn/Ag intensity increased as BSA volume increased. Blocking was complete when volume reached 600 μL (Fig. 4). Figure 4
Journal Pre-proof The volume and incubation times of MBs-Ab1-CYFRA21-1/CEA/CA15-3 were optimized to obtain high capture efficiency. This was achieved by fixing MBs-Ab1-CYFRA21-1/CEA/CA15-3 concentrations at 480 mg L-1 , and CYFRA21-1, CEA and CA15-3 concentrations at 50 ng mL-1 , 100 ng mL-1 and 250 mU mL-1 , respectively. CYFRA21-1, CEA and CA15-3 standard solutions with 10 μL was used. Ab2-CYFRA21-1-AuNPs, Ab2-CEA- ZnSe QDs, and Ab2-CA15-3-AgNPs volumes were 40 μL, 60 μL, and 60 μL, respectively. Incubation time was 1 h. The effect of MBs-Ab1-CYFRA21-1/CEA/CA15-3 volumes ranging from 20 μL to 80 μL on immunoreaction
197
Au/64 Zn/107 Ag increased rapidly with the increase of volume
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The number of detection events for
of
between MBs-Ab1-CYFRA21-1/CEA/CA15-3 and CYFRA21-1/CEA/CA15-3 was investigated.
for MBs-Ab1-CYFRA21-1/CEA/CA15-3 from 20 μL to 60 μL and then decreased from 60 μL to 80
-p
μL (Fig. 5a). A volume of 60 μL of MBs-Ab1-CYFRA21-1/CEA/CA15-3 was used to capture
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CYFRA21-1/CEA/CA15-3. Incubation time duration effects on immunoreactions between
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MBs-Ab1-CYFRA21-1/CEA/CA15-3 and CYFRA21-1/CEA/CA15-3 in the 20-120 min range was investigated. The results appear in Fig. 5b. The number of
197
Au/64 Zn/107 Ag detection events
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increased from 20 min to 60 min and remained constant after incubation for 60 min. This indicates immunoreaction completed in by the 60 min mark. An incubation time of 60 min was used in the
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following experiments.
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Figure 5
It was essential to add additional Ab2-CYFRA21-1-AuNPs, Ab2-CEA-ZnSe QDs, and Ab2-CA15-3-AgNPs to the reaction system. This was done by fixing Ab2-CYFRA21-1-AuNPs, Ab2-CEA-ZnSe QDs and Ab2-CA15-3-AgNPs concentrations at 22, 20 and 20 μg mL-1 , respectively.
The
effects
of
Ab2-CYFRA21-1-AuNPs,
Ab2-CEA-ZnSe
QDs
Ab2-CA15-3-AgNPs volumes were investigated for the 10 to 80 μL range. The number of
and 197
Au
detection events increased rapidly as Ab2-CYFRA21-1-AuNPs volume increased from 10 μL to 40 μL. It remained constant from 40 μL to 80 μL (Fig 6a). Forty μL of Ab2-CYFRA21-1-AuNPs was used. Similar results were obtained for Ab2-CEA-ZnSe QDs and Ab2-CA15-3-AgNPs. A volume of 60 μL of Ab2-CEA-ZnSe QDs and Ab2-CA15-3-AgNPs was used in the further experiments (Fig.
Journal Pre-proof 6a). The number of 197 Au/64 Zn/107 Ag detection events obtained at different incubation times ranging from 20 to 120 min appears in Fig. 6b.
197
Au/64 Zn/107 Ag detection events increased between 20 min
and 60 min, but remained constant after incubation for 60 min. This ind icates that the immunoreaction is completed within 60 min. A 60 min incubation time was used. Figure 6 3.5. Performance of analytical method
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Under the optimized experimental conditions, the analytical performance of the method was
ro
evaluated for simultaneous quantification of the three biomarkers. The plot between the number of detection events and CYFRA21-1/CEA/CA15-3 concentration appears in Fig. 7. The dynamic
-p
ranges were 0.05-50 ng mL-1 for CYFRA21-1, 0.02-100 ng mL-1 for CEA, 0.6-250 mU mL-1 for
re
CA15-3. Linear equations correlation coefficients were 0.9999, 0.9987, and 0.9970 for CYFRA21-1, CEA, and CA15-3, respectively. Our method’s detection limits were 0.02 ng mL-1 , 0.006 ng mL-1
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and 0.25 mU mL-1 for CYFRA21-1, CEA and CA15-3, respectively. The relative standard deviation (RSD) for the six replicate determinations was 3.2%, 4.4%, and 4.2% for CYFRA21-1, CEA and
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CA15-3, respectively. A comparison of the immunoassay methods for CYFRA21-1, CEA, and
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CA15-3 determination appear in Table S1. The detection limit of the present method for CYFRA21-1 is lower than previous immunoassays [7,8,32,33] and higher than that reported by
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Wang et al. [22]. The present method’s detection limit for CEA was lower than previous reports [7,8,22,32]. The detection limit of the present method for CA15-3 was lower than previous results [6,13,34,35]. Figure 7 3.6. Sample analysis The feasibility of this method was investigated. The concentrations of CYFRA21-1, CEA, and CA15-3 in human lung cancer serum samples (from the Guilin Fifth People’s Hospital (Guilin, China)) were tested by this method. For determination, serum samples spiked with standard CYFRA21-1, CEA, and CA15-3 were analyzed using a single particle mode ICP-MS. The results
Journal Pre-proof appear in Table 2. The recovery for CYFRA21-1, CEA and CA15-3 ranged from 89.8%-101%. Table 2
4. Conclusions In this work, we first reported an ICP-MS-based multiple immunoassay using AuNPs, AgNPs, and ZnSe QDs as element tags for simultaneous determination of CYFRA21-1, CA15-3, and CEA
of
in human lung cancer serum samples. Highly efficient captures of CYFRA21-1, CA15-3, and CEA
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were obtained using streptavidin- functionalized magnetic beads conjugated with a primary antibody.
-p
AuNPs, AgNPs, and ZnSe QDs-tagged second antibodies were used to determine the target antigen using SP-ICP-MS. The method was successfully applied to detect CYFRA21-1, CA15-3, and CEA
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in human lung cancer serum samples. This method is expected to have applications to other
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biomarker detection. Comparing to one element (Ag, Au, Zn), the simultaneous immunoassay of
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Acknowledgements
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multianalytes can improve assay efficiencies and reduce sample consumption.
This work was financially supported by the National Natural Science Foundation of China
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(21765004) and by the Guangxi Science Foundation of China (2019GXNSFAA245076), and by Guangxi University of Chinese Medicine Research Foundation (2018BS017). The research fund of State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources (Guangxi Normal University) (CMEMR2018-C18) is gratefully acknowledged.
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Journal Pre-proof List of Tables and Figures Table 1. ICP-MS instrument operating conditions. Table 2. Analytical results of human lung cancer serum samples (n=3). Scheme 1. Sandwich-type magnetic immunoassay using antibody-immobilized magnetic nanoparticles. Figure 1. Characterization of AuNPs and AgNPs. AuNPs and Ab2-CYFRA21-1-AuNPs conjugates UV-Vis spectrum (a). AgNPs and Ab2-CA15-3-AgNPs conjugates UV-Vis spectrum (b). AuNPs (c) and AgNPs (d) size
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distributions.
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Figure 2. Effects of Ab2-CYFRA21-1 amount (a) and Ab2-CA15-3 amount (b) on absorbance.
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Figure 3. Number of detection events per second and mean particle intens ity as a function of dwell time for
Figure
4.
BSA
blocking
volume
re
AuNPs (a) and AgNPs (b) dwell times.
effects.
MBs-Ab1-CYFRA21-1/CEA/CA15-3:
480
mg
L-1 ,
-1
-1
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Ab2-CYFRA21-1-AuNPs, Ab2-CEA-ZnSe QDs and Ab2-CA15-3-AgNPs concentrations at 22 μg mL , 20 μg -1
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mL and 20 μg mL , respectively. Incubation time was 1 h. Error bar represents s.d. value for triplicate analysis. Figure 5. MBs-Ab1 volume (a) and incubation time (b) effects on number of detection events. CYFRA21-1, CEA
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and CA15-3 standard solutions with 10 μL was used. Ab2-CEA-ZnSe QDs and Ab2-CA15-3-AgNPs
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concentrations at 22, 20 and 20 μg mL-1 , respectively. Error bar represents s.d. value for triplicate analysis. Figure 6. Labeled-Ab2 volume effects on the number of detection events (a) and incubation time effects on the number of detection events (b). MBs-Ab1-CYFRA21-1/CEA/CA15-3: 480 mg L-1 , CYFRA21-1, CEA and CA15-3 standard solutions with 10 μL was used. Error bar represents s.d. value for triplicate analysis. Figure 7. CYFRA21-1, CA15-3 and CEA calibration plot using SP-ICP-MS.
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Table 1. ICP-MS instrument operating conditions. Value
RF power (W)
1000
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Parameter
Nebulizer gas flow rate (L min-1 )
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Auxiliary gas flow rate (L min-1 )
-p
Plasma gas flow rate (L min-1 )
Scan mode
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RPq
0.9 1.20 15
Au, Ag: 0.5; Zn: 0.8 Peak hopping
Sample uptake rate (μL min-1 )
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7.5 0.1
Sweeps
1
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Dwell time (ms)
Cell gas flow rate DRC-CH4 (mL min-1 )
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Monitored isotope (m/z)
1.0 197
Au,
107
Ag,
64
Zn
Journal Pre-proof
(ng mL-1 )
(%)
(%)
16.9±0.08
91.8
3.5
14.6±0.10
89.8
4.3
10.0
9.88±0.05
98.8
3.8
15.0
27.0±0.05
95.3
4.2
6.73±0.08
10.0
16.2±0.03
94.7
4.0
---
10.0
10.1±0.12
101
3.1
18.3±0.05
20.0
37.3±0.09
95.0
3.8
CEA
8.11±0.05
10.0
18.1±0.05
99.9
3.8
CA15-3
1.19±0.03
2.00
2.99±0.02
90.0
3.5
Serum 2
(ng mL-1 )
7.72±0.05
10.0
CEA
5.62±0.15
CA15-3
---
CYFRA21-1
(male, 65 yrs)
12.7±0.07
CEA CA15-3 CYFRA21-1
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Serum 3
(ng mL-1 )
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(female, 66 yrs)
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Note: mU mL-1 for CA15-3
Total found
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(male, 56 yrs)
Added
10.0
-p
CYFRA21-1
Original
of
RSD
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Sample
Serum 1
Recovery
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Table 2. Analytical results of human lung cancer serum samples (n=3).
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Author Contribution Statement Yupin Cao: Conceptualization, Methodology, Investigation, Writing - Original Draft. Jinsu Feng: Visualization, Writing - Review and Editing. Lifu Tang: Data analysis, Writing – review & editing.
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Guichun Mo: Writing - Review and Editing. Weiming Mo: Visualization. Biyang Deng:
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Supervision.
Journal Pre-proof Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
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Highlights · The AuNPs, ZnSeQDs and AgNPs as elemental tags in sandwich-type immunoassay
· SP-ICP-MS was used to detect AuNPs, ZnSe QDs and AgNPs at the immunocomplexes
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· The proposed method succeeded simultaneous detection of CYFRA21-1, CEA and CA15-3
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· Total volume of immunocomplex solution was 10 μL for analysis.
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