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Application of spectral crosstalk correction for improving multiplexed microRNA detection using a single excitation wavelength Yuanjian Liu, Min Wei, Ying Li, Anran Liu, Wei Wei, Yuanjian Zhang, and Songqin Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04176 • Publication Date (Web): 01 Mar 2017 Downloaded from http://pubs.acs.org on March 2, 2017

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Analytical Chemistry

Application of spectral crosstalk correction for improving multiplexed microRNA detection using a single excitation wavelength Yuanjian Liu,† Min Wei,‡ Ying Li,† Anran Liu,† Wei Wei,*,† Yuanjian Zhang,† and Songqin Liu† †

Jiangsu Engineering Laboratory of Smart Carbon-Rich Materials and Device,

Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Chemistry and Chemical Engineering, Southeast University, Nanjing, 211189, China ‡

College of Food Science and Technology, Henan University of Technology,

Zhengzhou, 450001, China Phone: 86-25-52090613. Fax: 86-25-52090618. E-mail: [email protected]

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ABSTRACT MicroRNAs (miRNAs) play crucial roles in the regulation of cellular activities and are next generation biomarkers for early cancer detection. Simultaneous monitoring of multiplexed miRNA is very important for enhancing the accuracy of cancer diagnostics. Traditional fluorescence methods for multi-component analysis were usually operated under multiple excitation wavelength because spectral crosstalk are very detrimental to detection accuracy for multi-component analysis. Herein, we present a fluorescence strategy for multi-miRNAs detection in plasma under single excitation wavelength. Nucleic acid stain TOTO-1 and three labeled fluorescence dyes Cy3, Cy3.5 and Cy5 emit no fluorescence in their free state. Target miRNA hybridized the auxiliary and probe oligonucleotides into duplex nucleic acid. Intercalation interaction localized TOTO-1 and labeled dyes into the duplex nucleic acid. As a result, TOTO-1 emitted strong fluorescence and efficient Förster resonance energy transfer (FRET) happened. MiRNA-155, miRNA-182, and miRNA-197 that are significant for early diagnosis of lung cancer were simultaneously detected as models. Deviations from spectral crosstalk in the presence of other miRNAs were corrected by mathematical methods. Results demonstrated that after spectra crosstalk corrections every miRNA at high or low concentration in plasma were determined accurately in the presence of either high or low concentrations of the other two miRNAs. This new multiplexed assay for miRNAs is promising for clinical diagnosis, prognosis, and therapeutic monitoring of early stage lung cancer.

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Introduction MicroRNAs (miRNAs), a class of small noncoding endogenous RNAs, regulate gene expression at the posttranscriptional level by either repressing translation or decreasing the stability of target miRNAs.1-3 As miRNAs regulate over 50% of all human protein-coding genes,4 they play crucial roles in the regulation of cellular activities.5 Their distinct levels in serum, plasma, and other body fluids make them emerge as specific biomarkers to identify and define cancers at an early stage.6-12 For example, the level of miRNA-155, miRNA-182, and miRNA-197 always elevated when lung cancer happened.13 However, many cancer detection technologies were developed by using only a single miRNA as biomarker,7-9 which may induce false positive result for clinical diagnosis because of the insufficient information. This may cause anxiety for the individuals and lead to unnecessary biopsies or surgery. Compared with single-biomarker assays, multiplexed biomarkers assay not only enhanced the accuracy of cancer diagnostics14,15 but also offered several potential advantages in terms of improved detection efficiency, simplified analytical procedures, and reduced cost.16 Therefore, reliable detection of multiple biomarkers is an effective diagnostic strategy in the early stages of cancer. Actually, many efforts have been made to develop multiplexed analysis strategy. Surface-enhanced Raman scattering (SERS),17-19 microfluidic immunoarray,20 electrochemical,21 and fluorescent assays were prospect methods.22-24 Xu et al.17 used silver nanoparticles pyramids, which could bear multiple aptamers and triple Raman reporters, to develop multiple disease biomarker detection protocols. Otieno et al.20 described an ultrasensitive immunoarray to detect parathyroid hormone-related peptide and smaller peptide fragments using a novel semiautomated microfluidic device. Wu et al.21 described a novel electrochemiluminescence imaging platform for simultaneous detection of cancer biomarkers based on a closed bipolar electrode array. Förster resonance energy transfer (FRET),25 widely used for biological sensing due to its sensitivity and simplicity, possesses high prospects if used for multiplexed analysis. Noor et al. introduced a multiplexed solid-phase nucleic acid hybridization assay on a paper-based platform based on multicolor immobilized quantum dots (QDs).22 Green-emitting QDs and red-emitting QDs served as donors with Cy3 and Alexa Fluor 647 acceptors. Wang et al. developed a new and upgraded nanoflare, which

utilized

two-fluorophore-labeled

“flares”

for

3


ratiometric

fluorescent


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measurement of mRNA in living cells based on FRET.23 Zuo et al. developed a novel probe based on gold nanoparticles and polyA mediated diblock hairpin molecular beacons, which could be used for the multicolor detection of three different pancreatic cancer related miRNAs.24 Through analyzing the above detection technologies, conjugation of organic dyes to different biomolecules can be easily performed without significantly altering the biological functions of the fluorescent bioconjugates. However, fluorescence methods for multi-component analysis were usually operated under multiple excitation wavelength because spectral crosstalk are very detrimental to detection accuracy for multi-component analysis when single excitation wavelength were used. In order that the spectral overlap problems would be overcome, several novel approaches introducing a sophisticated spectral crosstalk correction have been developed recently. Qiu used multiplexed FRET between a luminescent Tb complex and three different QDs to sensitively detect three different miRNAs with a sophisticated spectral crosstalk correction, which resulted in very efficient background suppression.26 Jin et al. presented a fully homogeneous multiplexed miRNA FRET assay that combineed careful biophotonic design with a sophisticated spectral crosstalk correction.27 Geissler et al. also reported an optically multiplexed six-color FRET biosensor for simultaneous monitoring of five different individual binding events , which resulted in very efficient background suppression.28 Here, we present a novel strategy for detection of multiplex miRNAs simultaneously in plasma under single excitation wavelength. The method was based on FRET from nucleic acid stain TOTO-1 to fluorescence dyes including Cy3, Cy3.5, and Cy5 that were used as signal molecules. A spectral crosstalk correction was adopted to improve the detection accuracy. As a result, miRNA-155, miRNA-182, and miRNA-197 that are significant in the early diagnosis of lung cancer were simultaneously detected as models. A mathematical correction were used to diminish the signal interferences that come from the spectral corsstalk. Experimental Section Chemicals

and

Materials.

The

DNA-intercalating

dye

1,1′-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(be nzo-1,3-thiazole)-2-methylid-ene]-quinolinium tetraiodide (TOTO-1) was purchased from Thermo Fisher Scientific (Massachusetts, USA). 1×PBS (pH 7.2 ~ 7.4, 136.89 mM NaCl, 2.67 mM KCl, 8.24 mM Na2HPO4, 1.76 mM NaH2PO4) was diluted from 4


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a 10×DNAse/RNAase-free stock purchased from Sigma-Aldrich (St Louis, MO, USA). All other reagents of certified analytical grade were purchased from Sunshine Biotechnology (Nanjing, China). Ultrapure water (18.2 MΩ cm, Barnstead, Thermo Scientific) was used throughout the experiments. Cy3.5-functionalized oligomer was purchased from Eurogentec (Seraing, Belgium). Other labeled or unlabeled oligonucleotides were obtained from Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. (Shanghai, China). All oligomers were HPLC-purified, freeze-dried by the suppliers. The sequences of the oligomers were listed in Table S1. Fluorescence Property of TOTO-1 in Duplex Nucleic Acid Structure. The oligomers were diluted in ultrapure water to 1 µM as a stock solution. 5 µL of L1, P1′ and miRNA-155 were hybridized in 480 µL of 1×PBS at 25 °C for 120 min. Then, the assembled duplex oligomers were incubated with saturating amounts of TOTO-1 (at the ratio of 1 dye: 4 base pairs) for 60 min at 25 °C.29 TOTO-1 concentrations in duplex oligomers were determined according to its molar absorption coefficient ε = 117 000 M-1 cm-1.30 Procedures of Multiplexed miRNA Assay. For single-miRNA assay, duplex nucleic acid containing miRNA, fluorescence dye (Cy3, Cy3.5 and Cy5) labeled oligomers and auxiliary oligomers were skillfully designed (Table S2). MiRNA-155 was used as model to illustrate the operation of this detection: 10 nM L1 and 10 nM P1 in 500 µL 1×PBS incubated with various concentration of miRNA-155 ranging from 0 to 10 nM for 120 min at 25 °C. After incubation with 100 nM TOTO-1 for 60 min at 25 °C, the PL spectrums of TOTO-1 and corresponding fluorescence dyes were measured by fluorescence spectrometer. The spectral bandwidth for both excitation and emission monochromators was 5 nm. Samples were excited at 440 nm, and fluorescence was measured from 500 to 750 nm. The multiplexed miRNA assay was performed in homogenous solutions containing all three duplex oligomers structures. 10 nM L1, L2, L3 and 10 nM P1, P2, P3 in 500 µL 1×PBS with various concentration of three target miRNA ranging from 0 to 10 nM were hybridized at 25 °C for 120 min. After incubation with 100 nM TOTO-1, samples were excited at 440 nm, and PL intensities of three different fluorescence dyes were measured at 570 nm, 596 nm and 670 nm, respectively. Mathematical data processing was used to correct interferences from spectra overlap. Characterization. UV−vis absorption spectroscopy were obtained from an 5



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UV−vis spectrophotometer (Shimadzu UV−2450, Kyoto, Japan) at wavelengths ranging from 400 nm to 600 nm. All fluorescence measurements were conducted on a fluorescence spectrometer (Fluoromax-4, Horiba Jobin Yvon, Japan). A quartz cuvette QS 2 mm was used as sample container. The spectral bandwidth for both excitation and emission monochromators were 5 nm. Samples were excited at 440 nm, and fluorescence was measured from 500 to 750 nm. Energy transfer (ET) efficiency was determined by the decrease percentage of TOTO-1 in the presence of Cy3, Cy3.5, Cy5, respectively. Results and Discussion Characterization of TOTO-1 and Fluorescence Dyes of Cy3, Cy3.5, and Cy5. TOTO-1, a symmetric cyanine dye dimer, is a high affinity nucleic acid stain (Figure S1). They are nonfluorescent in the absence of nucleic acids. However, they exhibit strong fluorescent at 533 nm when they intercalates into duplex nucleic acids. Figure S2 illustrates the UV−vis absorbance spectra of TOTO-1 in the presence of duplex nucleic acid. The unbound dye has maximum absorbance at 514 nm and a weak absorbance at 480 nm. However, the situation was reversed in the presence of nucleic acids. Its absorbance at 480 nm was stronger than that at 514 nm. The reason was that the two chromophores in unbound dye were stacked into an “H”-dimer structure, however, nucleic acids separated the chromophores and restored the monomer spectrum.31 Fluorescence data of Cy3, Cy3.5, Cy5 and TOTO-1 were listed in Table S3. Figure 1A showed the absorption and emission spectrum of Cy3 (λmax,abs. = 550 nm, λmax,em. = 570 nm), Cy3.5 (λmax,abs. = 581 nm, λmax,em. = 596 nm), and Cy5 (λmax,abs. = 649 nm, λmax,em. = 670 nm). It can be seen that the emission peak of TOTO-1 overlapped the absorption peak of Cy3, Cy3.5, and Cy5 (Figure 1B). So, it was possible that TOTO-1 transferred their energy to the three fluorescence dyes simultaneously with single excitation, which was significant for multiplexed detection by using TOTO-1 as donor and Cy3, Cy3.5, and Cy5 as acceptors. More importantly, their maximum emission peaks were separated well from each other, which made it possible to detection miRNAs simultaneously under single wavelength excitation. The Principle of Multiplexed miRNA Assay. Lung cancer-relevant miRNA-155, miRNA-182, and miRNA-197 have high specificity and sensitivity to discriminate stage I lung cancer patients from cancer-free controls.32 In this work, we presented a novel strategy for the multiplexed detection of 6


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them in plasma based on FRET from TOTO-1 to three different fluorescence dyes. Mathematical data processing was used to obtain more real result by reducing the interferences from spectral crosstalk. Scheme 1 shows the principle of the multiplexed microRNA assay. Without target miRNAs, no duplex nucleic acid structure formed. TOTO-1 were in a free state and showed little fluorescence. Cy3, Cy3.5, and Cy5 were far away from the TOTO-1 under this circumstance, so, FRET was inhibited. In the presence of each target miRNA, it hybridized with fluorescence dye labeled and auxiliary oligonucleotides. Then, stable duplex nucleic acid structures formed respectively. As a result, light-harvesting dye of TOTO-1 intercalated into the duplex nucleic acid structure and emitted strong fluorescence. Under this circumstances, the confinement of TOTO-1 was in close proximity to three labeled dyes, resulting efficiently FRET from TOTO-1 to three fluorescence dyes. Thus, each concentration of miRNA could be detected based on its corresponding probes′ fluorescence intensity. Multiplexed miRNA assay. Single miRNA Assay. Firstly, the performance of single miRNA detection was investigated. Each dye was anchored in the duplex nucleic acid structure in order to obtain high FRET efficiency. Figure 2A shows that efficient FRET happened for every miRNA. The emission of TOTO-1 was quenched by 82%, 80%, and 85% for Cy3, Cy3.5, and Cy5, respectively. Taking into account the Förster distances of our system (see Supporting Information), such a high ET efficiencies cannot be explained by FRET between one donor and one acceptor. Energy migration among the cointercalated chromophores, ie., homo FRET among the TOTO-1 dyes, may be a possible reason.33 The PL intensities increased linearly with the increasing miRNA concentrations from 0.02 nM to 10 nM for all three miRNAs (Figure S4). Encouraged by the excellent performance of the single sensor assays we proceeded to investigate multiplexed miRNA detection. As shown in Figure 2B, the peaks of the corresponding dyes were separated well from each other when either two or three miRNAs were presented. These results demonstrated the feasibility of the multiplexed analysis of three miRNAs in the homogenous solution under single excitation wavelength. Spectral crosstalk correction for multiplex miRNAs assay. In single miRNA assay, PL intensities for Cy3, Cy3.5, and Cy5 were 8890, 9283, and 3872, respectively, in the presence of 10 nM corresponding target miRNA (Figure 2A). However, their PL intensities increased to 10923, 12284, and 5771, respectively (red curve in Figure 7



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2B) when the other two miRNAs (10 nM) were coexistent, indicating the spectral crosstalk existed under this circumstance. On the other hand, when Cy3 PL intensities increased with the increasing miRNA-155 concentration as that in single miRNA assay, the PL intensities for constant free concentrations of Cy3.5 and Cy5 also increased (Figure 3A). These performances just resulted from the spectral crosstalk between three fluorescence dyes, which was the main limitation for accurate quantitative determination in multiplexed biosensing. To overcome this drawback, a spectral crosstalk correction method was used for multiplexed miRNA detection.28 The spectral crosstalk between TOTO-1, Cy3, Cy3.5 and Cy5 were listed in Table 1. The values in the first column were obtained in comparison with the spectrum of Cy3 (green curve from Figure 2A), where the emission intensity of Cy3 is normalized to unity. As we know the intensity of Cy3 obviously decreases beyond its maximum at 570 nm, there is still significant spectral crosstalk contribution to the intensity of the other dye channels (36.8% to Cy3.5, 4.9% to Cy5). In this way, all the other dye columns could be quantified. What′s more, spectral crosstalk contribution of the dyes to the TOTO-1 channel intensity (last row in Table 1) can be neglected due to its much lower intensity compared to the TOTO-1 emission at this wavelength. The relation between the absolute intensities (I1−I3) arising from the emission of fluorescence dyes and the intensities emitted by each single dye (P1−P3) can be expressed by eq 1, where M is the 3×3 crosstalk correction matrix consisting of the first three columns and rows of Table 1. Matrix M can be inverted numerically in order to calculate I1−I3 from the measured absolute intensities P1−P3 via the highlighted part of eq 1. In order to obtain accurate results, the I1−I3 values must represent only FRET-sensitized acceptor emission. Consequently, any background signal must be subtracted prior to correction. The main background signal originated from fluorescence dyes. Fluorescence dyes will produce the weak fluorescence signals with 440 nm excitation without target miRNA (curve a in Figure S4). In order to achieve better sensitivity, this background contribution was subtracted prior to correction. I1 I2 I3

P1 = M· P2 P3

⇒

P1 P2 = M-1· P3

I1 I2 I3

(1)

8


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The obvious crosstalk of Cy3 (specific for miRNA-155) to other two dyes were observed in Figure 3A,B,C. After correction, the signals for the other two dyes remained at negligibly levels (Figure 3A′,B′,C′). The strong difference in uncorrected (I1−I3) and corrected (P1−P3) PL intensity values demonstrates the high efficiency of our correction algorithm, which allowed a precise calibration of Px to the target concentration. The Cy3 PL intensity increased monotonically with the logarithm concentration of miRNA-155 in the range of 0.02 nM to 10 nM with a detection limit of 18 pM based on the evaluation of average blank signal plus three times standard deviation (inset in Figure 3A′), which is superior to the obtained results without spectral crosstalk correction. The same correction method was also be used for assay of miRNA-182 and miRNA-197 in the presence of the other two dyes. Both of them had the same linear range as that for miRNA-155, and the detection limit for them were 12 pM and 11 pM, respectively (inset in Figure 3B′,C′), which was acceptable in comparison to previously reported methods (Table S4). In order to investigate the applicability of the biosensor in more complex conditions, the spectral crosstalk correction method was used to obtain more realistic results. In this section, we first measured the calibration curves of miRNA-155 when miRNA-182 was present at high, middle, or low concentrations. Figure 4A indicated that the calibration curves of miRNA-155 in the presence of 0.5 nM, 5 nM or 10 nM of miRNA-182 varied obviously. After the spectral crosstalk correction according to eq 1, the calibration curves were coincident well to each other in the presence of different concentrations of miRNA-182. The calibration curves for miRNA-182 and miRNA-197 in the presence of low, middle or high concentrations of another type of miRNA were also investigated by the same spectral crosstalk corrections (Figure 4B,C). Satisfactory results were obtained. The calibration curves varied little even in the presence of high concentrations of other miRNA targets. Multiplexed miRNA Assay in Plasma. Nine human plasma samples from S1 to S9 were diluted in a 1:10 ratio with 1×PBS. Every blank plasma sample were added into various concentrations of miRNA-155, miRNA-182, and miRNA-197. Concentration of each miRNA in every sample was skillfully designed according to Qiu’s method,26 which was indicated explicitly in Figure 5. The measured intensities of Ix were corrected to be Px via the eq 1. Then, the concentrations of each miRNA were calculated according to their corresponding calibration curves. Results were showed in Table 2. The obtained 9



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results after the spectra crosstalk correction were in well agreement with their known concentrations. Recoveries were from 90% to 109%, indicating that the method had good accuracy when it was applied in complex situations, for example, in the presence of three miRNAs at their low, medium, or high concentrations. Therefore, this multiplexed miRNA assay strategy based on FRET has great potential to be developed as a novel method for clinical diagnosis and evaluation of cancer development. Conclusions A novel strategy for the detection of multiplex miRNAs based on FRET from one nucleic acid stain TOTO-1 to three different organic dyes (Cy3, Cy3.5, and Cy5) using single excitation wavelength with a sophisticated spectral crosstalk correction was investigated. Target miRNA hybridized the auxiliary and probe oligonucleotides into duplex nucleic acid. Intercalation interaction localized TOTO-1 and labeled dyes into the duplex nucleic acid. As a result, TOTO-1 emitted strong fluorescence and efficient fluorescence resonance energy transfer (FRET) happened. MiRNA-155, miRNA-182, and miRNA-197 that are significant for early diagnosis of lung cancer were simultaneously detected as models. Deviations from spectral crosstalk in the presence of other miRNAs were corrected by mathematical methods. Nine human plasma samples added with various concentrations of miRNA-182, miRNA-197, and miRNA-155 were detected successfully with satisfactory results, indicating the accuracy and applicability of the proposed method.

Author Information Corresponding Author *Phone: 86-25-52090613. Fax: 86-25-52090618. E-mail: [email protected]. Notes The authors declare no competing financial interest. Acknowledgments This work was financially supported in part by the National Natural Science Foundation of China (Grant Nos. 21475020 and 21375014), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (Grant Nos. 1107047002), and the Fundamental Research Funds for the Central Universities. Supporting Information 10


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The sequences of the oligomers, fluorophore photophysical and FRET properties, optimization of TOTO-1 concentration and incubation time were listed in Supporting Information.

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Figure captions Scheme 1. Principle of the multiplexed miRNAs assay in homogeneous solution under single wavelength excitation based on FRET.

Figure 1. (A) Normalized absorption/emission spectra for fluorescence dyes used in the manuscript. (B) PL emission spectra of TOTO-1 donor (black curve with gray background) and absorbance spectra of the acceptor dyes: Cy3 (green curve), Cy3.5 (orange curve), and Cy5 (red curve), respectively.

Figure 2. (A) PL spectrums of TOTO-1 that intercalated in duplex nucleic acid, Cy3, Cy 3.5, and Cy5 in the presence of its corresponding miRNA-155, miRNA-182 and miRNA-197, respectively. (B) PL spectrums in the presence of two miRNA targets or three miRNA targets. Spectra acquired by excitation at 440 nm. Samples contained 10 nM corresponding miRNA structure model and 100 nM TOTO-1 in 1×PBS.

Figure 3. Single miRNA assay results for miRNA-155 (A), miRNA-182 (B), and miRNA-197 (C) concentrations of 0−10 nM in the presence of all probes before (right) and after spectral crosstalk correction (left) using eq 1. Inset: calibration curves for corresponding miRNA. The regular triangle (▲), dots (●), and inverted triangle (▼) represent the Cy3, Cy3.5, and Cy5 channel intensities, respectively. Samples contained 10 nM fluorescence dye labeled oligomers, 10 nM auxiliary oligomers and 100 nM TOTO-1 in 1×PBS with corresponding miRNA at different concentrations.

Figure 4. Calibration curves of miRNA-155 (A), miRNA-182 (B), and miRNA-197 (C) in the presence of high (10 nM, curve a, a′), middle (5 nM, curve b, b′) or low concentrations (0.5 nM, curve c, c′) of miRNA-182, miRNA-197, and miRNA-155, respectively. a, b, and c represent calibration curves before corrections, while a′, b′, and c′ represent calibration curves after spectral crosstalk correction.

Figure 5. Nine human plasma samples were added into three different concentrations of miRNA. Regular triangles, inverted triangles and dots represent the concentration of miRNA-155, miRNA-197 and miRNA-182, respectively.

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Scheme 1

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Table 1. Normalized spectral crosstalk intensity contributions of the different fluorophores measured under single excitation wavelength.a detection channel

a

Cy3

Cy3.5

Cy5

TOTO-1

Cy3 (570 nm)

1.000 ± 0.012

0.215 ± 0.027

0.000 ± 0.011

0.015± 0.002

Cy3.5 (596 nm)

0.368 ± 0.016

1.000 ± 0.008

0.005 ± 0.010

0.005± 0.001

Cy5 (670 nm)

0.049 ± 0.018

0.157 ± 0.017

1.000 ± 0.004

0.000± 0.001

TOTO-1 (533 nm)

0.005 ± 0.015

0.000 ± 0.005

0.000± 0.001

1.000 ± 0.017

The highlighted part is the 3×3 crosstalk correction matrix M (cf. eq 1).

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Table 2. Recovery results of miRNA added in human plasma samples (volume ratio 1:10, plasma/1×PBS).b

sample no. S1 S2 S3 S4 S5 S6 S7 S8 S9

added (nM) 0.5 1 3 7 10 10 10 10 10

miRNA-155 found recovery (nM) (%) 0.45 1.08 2.93 6.89 9.71 10.3 9.85 10.2 10.9

90.0 108 97.7 98.4 97.1 103 98.5 102 109

added (nM)

miRNA-182 found recovery (nM) (%)

10 7 3 1 0.5 1 3 7 10

9.91 6.89 2.86 0.95 0.47 1.15 3.24 7.11 10.3

99.1 98.4 95.3 95.0 94.0 115 108 102 103

b

added (nM) 10 10 10 10 10 7 3 1 0.5

miRNA-197 found recovery (nM) (%) 10.1 10.5 9.95 9.84 10.2 6.65 2.91 0.98 0.45

101 105 99.5 98.4 102 95.0 97.0 98.0 90.0

Human serum samples are sampling from healthy donors at the Second Affiliated Hospital of Southeast University, Nanjing, China.

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for TOC only

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Application of Spectral Crosstalk Correction for ... - ACS Publications

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