Evaluation of Multidrug Resistance of Leukemia Using Surface

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Biological and Medical Applications of Materials and Interfaces

Evaluation of Multidrug Resistance of Leukemia Using SERS Method for Clinical Application Yujie Wang, Shenfei Zong, Lei Wu, Yizhi Zhang, Zhile Wang, Zhuyuan Wang, Baoan Chen, and Yiping Cui ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02917 • Publication Date (Web): 12 Jul 2018 Downloaded from http://pubs.acs.org on July 12, 2018

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ACS Applied Materials & Interfaces

Evaluation of Multidrug Resistance of Leukemia Using SERS Method for Clinical Application Yujie Wanga, b, Shenfei Zonga, Lei Wua, Yizhi Zhanga, Zhile Wanga, Zhuyuan Wanga*, Baoan Chenb*, Yiping Cuia* a

Advanced Photonics Center, Southeast University, Nanjing 210096, Jiangsu, China

b

Department of Hematology and Oncology, Zhongda Hospital, School of Medicine,

Southeast University, Nanjing 210009, China * [email protected], [email protected], [email protected]

Abstract P-glycoprotein (P-gp) is an important multidrug resistance (MDR) regulator for leukemia to mediate its development, thus can be considered as a powerful reference for the diagnosis of MDR. The detection of P-gp is of vital significance and has attracted considerable concerns. In this study, we proposed a surface enhanced Raman scattering (SERS) method for the evaluation of P-gp expression levels in leukemia cell lines. Basically, we utilized an aqueous phase sandwich-type immunoassay to analyze the expression of P-gp. First, anti-CD45 decorated magnetic beads (MBs) and P-gp antibody decorated SERS probes were fabricated. CD45 is a common protein expressed in all leukemia cells. As a result, a sandwich immunocomplex can be formed by the MBs, P-gp overexpressed leukemia cells and SERS probes. The expression level of P-gp determines the amount of SERS probes that can be captured. Consequently, the SERS intensity of the immunocomplex can be used to evaluate the expression level of P-gp. In a typical procedure, we measured the P-gp expression of a MDR leukemia cell line (K562/ADM) as well as unprocessed whole-blood samples. The SERS intensity of K562/ADM cells was highly correlated with the extent of MDR or the incubation time of adriamycin (which is a MDR inducing drug). In addition, the SERS intensity of the 1

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refractory/relapsing group was about six folds of that of the control groups (P＜0.01). These results demonstrated that the proposed method holds excellent sensitivity, specificity, reliability and application potential in assessing both cultured cells and clinical samples. With these outstanding features, we anticipated that such a SERS based method could be very helpful for the clinical diagnosis of early-stage MDR in leukemia. Keywords: SERS, P-glycoprotein (P-gp), multidrug resistance (MDR), leukemia, clinical application 1. Introduction Leukemia is one of the hematologic malignancies that severely threaten human health and lead to economic burdens. There is a significant prognostic difference between primary and refractory/relapsing patients.1-2 The most important reason is that the survival rates of refractory/relapsing patients decrease due to the development of multidrug resistance (MDR) in the chemotherapy of leukemia.3 One of the best recognized mechanisms of MDR in leukemia is mediated by P-gp, which is produced by the MDR protein 1 (MDR1) gene and acts as a pump expelling drugs out of the cell.4 Thus MDR1 gene and P-gp are considered as representative and significant indicators for MDR assessment in clinical settings.5-6 Senior physicians usually design chemotherapy strategies according to the P-gp expression level. A large number of reports have shown that overexpression of P-gp will reduce the complete remission (CR) time and overall survival.3, 7 To analyze the expression level of P-gp, some traditional methods, such as flow cytometry (FCM), western blotting (WB), reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH), have been widely used in biomedical research and clinic diagnosis.8-9 FCM and WB are easy to grasp, but they are time-wasting and have limited sensitivity. RT-PCR and FISH are relatively novel techniques with higher sensitivity, whereas their clinical application is limited due to relatively higher costs. Therefore, we designed a SERS based method to detect the 2


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expression level of P-gp in MDR leukemia cells, aiming to realize diagnosis of MDR with higher sensitivity, specificity, simplicity and applicability. In recent years, SERS has emerged as a new detection technology of biological molecules with the advantages of ultrasensitive screening ability and extensive adaptability.10-13 SERS, as a non-invasive detection technology, can provide abundant information about the material composition and molecular structures. As one representative application, SERS-based immunoassays can differentiate cancerous and normal tissues at a molecular level, hence, they are used for accurate cancer diagnosis.14-15 As for SERS based applications in leukemia research, Nguyen et al. used SERS nanoparticles to recognize chronic lymphocytic leukemia cells by identifying CD19 antigen on the surfaces of leukemia cells.16 Yu et al. employed an electroporation-based SERS technique for high-throughput analysis of leukemia cells and reached a diagnostic accuracy as high as 96.7%.17 For MDR detection, Krishna et al. utilized micro-Raman spectroscopy to distinguish sensitive and resistant leukemia and breast cancer cell lines without target biomarkers.18 Later they also used the micro-Raman spectroscopy to investigate a sensitive human uterine sarcoma cell line and its MDR product with high accuracy and sensitivity. To date, SERS technology has been used for the detection of clinical samples, such as detection of multiple prostate cancer markers in serums,19 exosomal microRNAs extracted from human blood,20 and circulating tumor cells (CTCs).21 However, few researches have focused on the application of SERS immunoassays for the diagnosis of MDR in leukemia patients, especially when using the unprocessed whole-blood samples. As a result, we presented here a SERS-based method to detect the P-gp expression levels in MDR leukemia cells (K562/ADM) and unprocessed whole blood collected from patients. To be specific, we developed a sandwich immunoassay protocol, in which anti-CD45 decorated MBs are used as the immuno-substrates and anti-P-gp decorated SERS probes are used as the immuno-probes. Since the K562/ADM cells express CD45 3



and P-gp on their membranes, immunocomplexes would be formed by the MBs, SERS probes, and K562/ADM cells through immuno-reactions. The immunocomplexes could be precipitated by a magnet and then the intensity of SERS signals was detected, which can be used to indicate the presence and amount of K562/ADM cells (Scheme 1). To perform dynamic and qualitative analysis of the occurrence of drug-resistance, we used K562/ADM cells as a representative MDR cell line and K562 cells as a representative drug-sensitive cell line. By mixing these cell lines with different concentrations, a MDR model was established to simulate the occurrence of MDR. Then, the SERS based method was used to analyze the MDR model. Moreover, to investigate the feasibility of our proposed assay as a clinical tool in the diagnosis of MDR leukemia, whole blood samples from 45 leukemia patients were detected. The results of SERS based detection were compared with those of FCM assay and they were highly consistent. The experimental results proved that the proposed SERS immunoassay could provide an accurate diagnosis of MDR by simply measuring the P-gp expression level. With the advantages of high sensitivity, reduced sample consumption, and rapidness, the SERS based method is a powerful tool for monitoring the chemotherapy procedures of leukemia patients. 2. Materials and Methods 2.1. Cell culture and MDR model preparation Leukemia cells (K562 and K562/ADM) were obtained from Institute of Hematology, Chinese Academy of Medical Sciences. Another two drug-sensitive leukemia cells (i.e. HL-60 cells and U937 cells) with low-expression level of P-gp were also used. U937 cells were obtained from Nanjing Medical University, HL-60 cells were purchased from the Cell Bank of the Chinese Academy of Sciences. Cell culture and MDR maintenance were performed as described before.22 To establish MDR models of various drug resistance levels and simulate the MDR 4


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occurrence, K562 and K562/ADM cells were mixed with different amounts. The ratios between K562 and K562/ADM cells were 0:1, 0.25:0.75, 0.5:0.5, 0.75:0.25, 1:0, respectively. To assess the sensitivity of the SERS method, different concentrations of K562 and K562/ADM cells were used for the quantitative analyses, which were 5×106, 5×105, 5×104, 5×103, 5×102, and 50 cells/mL, respectively. 2.2. Cell Counting Kit-8 assay The viability of leukemia cells was evaluated by a CCK8 assay. After seeding the cells in 96-well plates at a density of 5×105 cells/mL, K562 and K562/ADM cells were incubated with different concentrations of adriamycin (i.e. the drugs) for 24 h. The cytotoxic effect of adriamycin was determined by CCK8 assay. The optical density at 450 nm (OD 450) was recorded. 2.3. Whole-blood samples 45 clinical blood samples from acute leukemia patients were obtained from Zhongda Hospital, Affiliated to Southeast University, China. These patients were diagnosed by senior physicians, by checking their bone marrow cell morphology and pathology. The clinical types of these patients were determined according to the World Health Organization (WHO) classification.23 Whether the patients were primary or refractory/relapsing was distinguished on the basis of National Comprehensive Cancer Network (NCCN) clinical practice guidelines.24-26 The expression levels of P-gp of all the samples were detected by FCM. The result (shown in percentage) is defined by dividing the number of P-gp positive cells with the whole number of cells. According to the studies of UT MD Anderson Cancer Center, if the result is more than 10%, it means the sample overexpresses P-gp. If the result is less than 10%, it indicates the sample has a low-expression level of P-gp. The 45 blood samples were divided into three groups, Group A (primary patients), Group B (refractory/relapsing patients with low-expressed P-gp), and Group C (refractory/relapsing patients with over-expressed P-gp). The 5



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demographic statistics and clinical characteristics were shown in Table S1. Our study received ethics approval and no-informed consent from the ethics committee of Zhongda Hospital,

Affiliated

to

Southeast

University

(2016ZDSYLL018.1).

Different

concentrations of K562/ADM cells were also added into healthy human blood for qualitative analysis, ranging from 5×106 to 50 cells/mL. All cell suspensions and blood samples were stored at 4℃. 2.4. Fabrication of the SERS probes and magnetic nanobeads The MBs (Scheme 1A) and SERS probes (Scheme 1B) were fabricated as described in our previous publications.27 Briefly, Ag nanoparticles (Ag NPs) were first synthesized according to a previously published method.27 Then Ag NPs were mixed with 4-mercaptobenzoic acid (4-MBA) ethanol solution and coated with silica shells using Stöber’s method,28 forming the Ag@4-MBA@SiO2 NPs. Similarity, silica coated MBs (MB@SiO2) were produced. Afterwards, MB@SiO2 and Ag@4-MBA@SiO2 NPs were decorated with CD45 and P-gp antibodies on their surfaces respectively using glutaraldehyde as the linker.29 All the experiments were conducted at room temperature. 2.5. Sandwich immunoassay The cells and whole blood samples prepared above were the target samples in our experiments. 100 µL targeted samples were added into 100 µL solution containing SERS probes (1:3000 in 1×PBS) and magnetic nanobeads (1:5000 in 1×PBS). After shaking at room temperature for 1.5 h to allow immuno-reactions, the sandwich immunocomplexes, which were comprised of the SERS probes, target samples, and MBs, were collected by a magnet and then suspended in 10 µL deionized water (Scheme 1C, D). To confirm that MBs can successfully capture the target cells, another experiment was also conducted, where cells were first stained with NucRed Live 647 (a nucleus dye). Then, after immuno-reaction with MBs, fluorescence imaging of the precipitates were performed with a confocal laser scanning microscope. All the experiments were conducted at room 6


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temperature. 2.6. SERS measurements Typically, 2 µL of each immunocomplexes corresponding to different target samples were pipetted onto a glass slide. After gradual evaporation of the water, another 2 µL of the identical immunocomplexes was pipetted onto the exact same position on the glass slides. The procedure, which can concentrate the samples, was repeated for 5 times before SERS measurements. Each SERS measurement was repeated three times to obtain average results. All the experiments were operated at room temperature.

3. Result and Discussion The concept of the proposed SERS-based method for detecting MDR of leukemia is demonstrated in Scheme 1. First, the formation of a sandwich immunocomplex was driven by the specific interaction between the corresponding antigen and antibodies. Specifically, anti-CD45 antibodies decorated MBs could capture the target samples. Meanwhile, the SERS probes could also bind to the target cells through the interaction between the P-gp antibodies and P-gp molecules on the target samples. With more P-gp presented, more SERS probes would be captured and thus stronger SERS signals would be detected. Hence, the SERS intensity can be used to evaluate the expression level of P-gp on the membranes of target cells. 3.1 Characterization of SERS probes and MBs Figure S1 shows the TEM images of Ag NPs and the Ag@4-MBA@SiO2NPs. The Ag NPs show a mean diameter around 30 nm and the silica shell is approximately 35 nm thick. The TEM images of Fe3O4 MBs before and after being coated with a layer of silica are shown in Figure S2. The size of the Fe3O4 MBs varies from 100 to 200 nm and the average thickness of the silica shell is about 50 nm. In order to verify that the target cells can be successfully captured by MBs through the 7



anti-CD45 antibodies, the K562/ADM cells were stained by fluorescent nuclear dyes as described in Section 2.5. Then, the stained cells were incubated together with SERS probes and MBs to form sandwich immunocomplexes. SERS probes and MBs incubated with only PBS buffer (without target cells) were used as the control. After immuno-reaction, the fluorescence images of the precipitates were taken by a confocal microscopy and the results are shown in Figure S3. A large number of red spots were observed in Figure S3A, which indicated the presence of K562/ADM cells. On the contrary, for the control sample without target cells (Figure S3B), we could barely find any red spots. These results confirmed that K562/ADM cells could be successfully captured by MBs. The sparse red spots in Figure S3B might be due to some nonspecific adsorptions, although we have used BSA to block SERS probes and MB@SiO2. Furthermore, to confirm that P-gp molecules on the surfaces of the target cells could be precisely recognized by the SERS probes, we also performed fluorescence cell imaging experiments of the precipitates, using SERS probes decorated with FITC-labeled P-gp antibodies to recognize the cells in the precipitates. As shown in Figure S4, the sample contained K562/ADM cells showed strong fluorescence signals while the one with K562 cells exhibited weak signals. Besides, there was no detectable fluorescence signal from the negative control sample using only PBS solution (without cells). The above results indicated the successful recognition between P-gp antigens on the cell surfaces and their antibodies on the SERS probes. In addition, to validate that sandwich immunocomplexes did form, the precipitates obtained from the experiments were subjected to TEM imaging. The TEM image (Figure S5) showed that the K562/ADM cells (red circles and arrow) were surrounded by the SERS probes (yellow circles) and MBs (blue circles), suggesting a complete sandwich-type structure. 3.2 Diagnosis of MDR in leukemia cell lines using the SERS method Before the experiment, we first evaluated the IC50 value of adriamycin (the chemotherapy drug) by a CCK8 assay. The IC50 value, demonstrating the drug-resistance 8


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ability of K562/ADM cells, is a representative value of the drug concentration that is required to inhibit 50% of the cell viability. Based on the results from the dose–response of CCK8 assay (Figure S6A), we calculated that the IC50 value of K562/ADM cells was 7.22 µg/mL, which was about 34 times more than that of K562 cells (0.21 µg/mL). This result suggested that the resistance against adriamycin in K562/ADM cells was apparent and thus K562/ADM cells can be qualified as a MDR leukemia cell line. In conclusion, the IC50 curves provided a convincing evidence for the following in vitro cell experiments using K562/ADM as the MDR cells. Next, we carried out a series of MDR measurements using the SERS immunoassay. To evaluate the precision of this immunoassay, first, SERS measurements were performed using K562 cells and K562/ADM cells with the same cell concentration (i.e. 5×106 cells/mL). In addition, another two kinds of drug-sensitive leukemia cells with low-expression level of P-gp (HL-60 cells and U937 cells) were also tested for comparison. As indicated in Figure S6B, the intensity of SERS signals of K562/ADM was much higher than those of the other three cell lines, indicating that the SERS probes could specifically recognize MDR cells. The results in Figure S6B confirmed that the SERS immunoassay can selectively and accurately detect MDR of leukemia. In the study, different ratios of K562 and K562/ADM cells were used as the model for different MDR levels. During the experiments, the total cell amount was kept the same, the only difference is the ratio between K562 and K562/ADM cells. When the K562 cells were mixed with K562/ADM cells by different ratios (0:1, 0.25:0.75, 0.5:0.5, 0.75:0.25, 1:0), the intensities of SERS signals exhibited a good linear response to the ratios (R2=0.988) (Figure 1). As can be seen, with larger ratio or amount of K562/ADM cells, stronger SERS signals were detected. Since in such a MDR model, larger ratio of K562/ADM means a higher MDR level, consequently, the SERS intensity can be used to monitor the MDR levels. Hence, the proposed SERS based method can indeed be employed to analyze MDR in leukemia cell lines. 9



We also investigated the detection sensitivity of this SERS-based method. In the experiments, K562/ADM cells of different concentrations ranging from 5×106 to 50 cells/mL were prepared for SERS measurements while pure PBS solution without cells was used as a control. These cells with different concentrations were subjected to SERS immunoassay. The results are shown in Figure 2A and B. As can be seen, when the cell concentration decreased, the intensities of SERS also exhibited a linear declining trend (R2=0.991). The weak SERS signal of the PBS control originated from nonspecific adsorptions. Meanwhile, K562 cells of different concentrations ranging from 5×106 to 50 cells/mL were also tested and the results are shown in Figure 2C and D. Similarly, the intensities of SERS were linearly correlated to the concentrations (R2= 0.981). The average intensity of SERS signals of K562 cells was much weaker than that of K562/ADM cells. This is rational because with less P-gp presented, fewer SERS probes were captured, resulting in weaker SERS intensity. As for clinical samples, the number of white blood cells in human blood normally falls between 4×106 to 10×106 cells/mL. After chemotherapy, the white blood cells of leukemia patients would decrease to an extremely low level (0.1×106 to 1×106 cells/mL), but it is still within the dynamic range of this assay. So our SERS immunoassay has a great potential in clinical leukemia MDR detection. After confirming the specificity and sensitivity of the SERS method, the ability of this method to monitor the dynamic occurrence of drug-resistance in leukemia cells was investigated. Usually, a high dose of adriamycin can quickly stimulate the resistance of leukemia cells, resulting in an increased expression level of P-gp on the membranes of the cells. Hence, in our experiments, K562/ADM cells were treated with adriamycin in two different ways to generate dynamic MDR models. In the first experiment, K562/ADM cells were incubated with adriamycin of different concentrations (0, 2, 4, 8 and 16 µg/mL) for 1 h. Afterwards, the K562/ADM cells with different expression levels of P-gp proteins were subjected to SERS detections. As shown in Figure S7A, the intensity of SERS signals increased significantly when K562/ADM cells were incubated 10


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with increased doses of adriamycin. In other words, the intensity of SERS signals would increase with the enhanced resistance degree of K562/ADM cells. In the second experiment, K562/ADM cells were incubated with 10 µg/mL of adriamycin for different times (0, 30, 60, 90 and 120 min). The SERS immunoassay results were shown in Figure S7B, which demonstrated that the intensity of SERS signals increased when the cells were incubated with adriamycin for longer time. When the incubation time reduced to zero or without any drug incubation, SERS signals were still observed. This is because K562/ADM cells are originally drug-resistant and P-gp overexpressed cells. In a word, incubation with the drug of a higher concentration or longer time could largely strengthen the level of drug-resistance. The results of the above two experiments both strongly proved that the proposed SERS immunoassay could dynamically monitor the degree of MDR in leukemia cells. 3.3. Analyzing whole blood samples collected from patients Before testing whole blood samples, we investigated the signal stability of the SERS probes suspended in blood for different time periods (ranging from 0 to 120 min). We found that the SERS signals remained stable for different times (Figure S8), which means the SERS probes can be used for blood samples. MDR diagnosis plays important roles in the chemotherapy of leukemia, thus a rapid, accurate, simple and cheap method for the detection of P-gp is vital. Though FCM of bone marrow is the most widely used method in clinic for P-gp detection, it has obvious limitations due to its complicated operation procedures. Considering that SERS-based methods only require a tiny amount of samples, simply taking several drops of blood for the SERS detection could be acceptable for the patients as a noninvasive assessment. Here, to investigate the potential and reliability of this SERS immunoassay in clinical applications, two different experiments were performed. In the first experiment, blood samples from healthy people were mixed with different amounts of K562/ADM cells 11



(from 5×106 to 50 cells/mL) and analyzed using the SERS immunoassay. The results were shown in Figure 3A and B, which confirmed the sensitivity of our SERS immunoassay in real blood samples. One thing needs to be mentioned here is that, if we compared this result with the one of K562/ADM cells in PBS (Figure 2A and B), the same concentrations of K562/ADM cells in blood samples showed a slightly weaker SERS signals. It is reasonable because there were not only K562/ADM cells in the blood but also normal cells. So the actual concentrations of MDR K562/ADM cells were lower than those of pure K562/ADM cells, which resulted in a decreased SERS intensity. In the second experiment, the P-gp expression levels of 45 unprocessed blood samples divided into three groups were directly subjected to SERS measurements (Figure 4). As shown in Figure 4D, the average SERS intensity in group C was almost six times higher than those of Group A and group B (P＜0.01), indicating that MDR cells in whole blood samples could be successfully detected without the interference of other cells or impurities. Finally, the correlation between FCM and SERS measurement was analyzed (Figure 4A-C). A high consistency between the two methods was observed (P=0.49), proving that our SERS immunoassay was reliable for unprocessed blood samples and had a good potential in the clinical applications of MDR diagnosis.

Conclusion A SERS-based immunoassay method has been developed to measure the expression level of P-gp (a biomarker for MDR) for the diagnosis of MDR in leukemia. The specificity of the immunoassay was verified by monitoring the SERS intensity of the drug-resistant cells with different expression levels of P-gp. Quantitative analysis of P-gp using both pure K562/ADM cells and blood mixtures also showed outstanding sensitivity. More importantly, for clinical applications using unprocessed blood, the SERS immunoassay also displayed great reliability when distinguishing patients with MDR. All these features demonstrate that the SERS-based approach is a sensitive and convenient technique for the 12


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diagnosis of leukemia MDR, especially in clinical applications. The present study shows a guiding significance in the treatment and prognosis of refractory/relapsing leukemia patients, thus it holds excellent potential in improving the survival rate and clinical outcomes of leukemia patients.

Supporting information The supporting information contains details of the materials, instruments, and statistical analysis, the supporting table (Table S1), and supporting figures (Figures S1-S8).

Competing Interests The authors have declared that no competing interest exists.

Acknowledgments This work was supported by the Natural Science Foundation of China (NSFC) (Grant No.61535003), the National Key Basic Research Program of China (Grant No. 2015CB352002), the Natural Science Foundation of China (NSFC) (Grant No. 61675042 and 61505027), the Fundamental Research Funds for the Central Universities (2242018K3DN11) and the Key Department of Jiangsu Province (ZDXKB2016020).

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Scheme 1. Illustration of the SERS-based immunoassay. (A) Synthesis of the MBs. (B) Synthesis of the SERS probes. (C) Sandwich immunocomplex formed by the magnetic nanobead, target cell and SERS probe. (D) Enrichment of the sandwich-type immunocomplexes by a magnet.

Figure 1. (A) SERS spectra corresponding to different ratios of cells. (B) Linear regression of the peak intensity at 1078 cm-1and different ratios of cells. The calibration equation is y=1333.6-2330.7x (n=3, R2=0.988).

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Figure 2. (A) SERS spectra of K562/ADM cells with different concentrations. The concentration of K562/ADM cells ranges from 50 to 5×106 cells /mL. Pure PBS without cells was used as the blank control. (B) Linear regression of the peak intensity at 1078 cm-1 and the concentration of K562/ADM cells. The calibration equation is y=-1821.7+2034.5x (n=3, R2=0.991). (C) SERS spectra of K562 cells with different concentrations. The concentration of K562 cells ranges from 50 to 5×106 cells /mL. Pure PBS without cells was used as the blank control. (D) Linear regression of the peak intensity at 1078 cm-1 and the concentration of K562 cells. The calibration equation is y=-70.45+429.5x (n=3, R2=0.981).

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Figure 3. (A) SERS spectra of K562/ADM cells added in blood sample. The concentration of K562/ADM cells ranges from 50 to 5×106 cells /mL. 100 µL PBS added into 1 mL human blood without any leukemia cells was used as the blank control. (B) Linear regression of the peak intensity at 1078 cm-1 and the concentrations of K562/ADM cells. The calibration equation is y=-570.5+1662.4x (n=3, R2=0.983). (C) SERS spectra of K562 cells added in blood sample. The concentration of K562 cells ranges from 50 to 5×106 cells /mL. 100 µL PBS added into 1 mL human blood without any leukemia cells was used as the blank control. (D) Linear regression of the peak intensity at 1078 cm-1 and the concentration of K562 cells. The calibration equation is y=218.4+422.6x (n=3, R2=0.989).

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Figure 4. Passing−Boblok regression analyses between FCM and parallel SERS measurements in three different groups of patients: (A) primary patients, (B) refractory/relapsing patients with low-expressed P-gp, (C) refractory/relapsing patients with over-expressed P-gp. (D) Box plots of the peak intensity at 1078 cm-1 corresponding to the clinical whole blood samples of the three groups.

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