Malignant tumors are common and lethal [1–3]. Recurrence and metastasis are the two main problems with cancer therapy [4]. Currently, treatments for cancer can be separated into three categories: radiation, surgical removal of a tumor, and chemotherapy [5,6]. However, the cancer can recur at or near the site of the tumor or distant points [7]. Cancer recurrence can be a combination of various factors. One of the main causes is that incomplete cancer treatment results in the surviving of cancer cells in the patient’s body.

The early diagnosis and adjuvant treatment could improve the chance of killing any remaining cancer cells and reduce the risk of relapse. Early detection of recurrence during the follow-up is associated with improved survival rates in patients with early-stage cancer [8] regardless of whether they have received adjustment therapy [9]. The biomarker now is used as the standard of cure, in which a carcinoembryonic antigen (CEA) [10] has limited sensitivity. Computed tomography (CT) [11] imaging improves the detection of recurrences, but it is associated with radiation exposure and also a high false-positive rate.

Nanomedicine aims to improve the biodistribution and the target site accumulation of systemically applied chemotherapeutics. Organic dyes [12], nanoparticles [13], semiconductors [14], quantum dots [15], and other nanomaterials [16] have been extensively investigated. These systems offer a novel combination of tunable photoluminescence, excellent photostability, biocompatibility, and chemical inertness. Moreover, many routinely used (chemo)therapeutic agents suffer from poor pharmacokinetics and biodistribution. Because of their low molecular weight, for instance, intravenously administered anticancer drugs are generally rapidly cleared from the circulation (by means of renal filtration), and they do not accumulate well in tumor cells. Strong photobleaching and inferior thermal stability of conventional organic dyes as well as the cytotoxicity of nanoparticles and semiconductor quantum dots hinder practical applications. The detection of an abnormal chromosome number in cancer cells [16–19] is a signal to successful cancer treatment. Normal cells are diploid, and the cancer cell is nondiploid because of spontaneous mitotic chromosome nondisjunction in the cancer cell [16]. Different intensities of fluorescence can be detected by optical techniques due to the different chromosome number.

In this paper, we provide a fluorescence detection map drawn by a strong coupling waveguide, hollow-core metal-cladding optofluidic waveguide, to detect the intensity of fluorescence from a single cell and analyze the chromosome number in the cell. Our strategy is described below. First, cells with DAPI-stained chromosomes are injected into a sample channel one by one. Meanwhile, the specific ultrahigh-order modes (UOMs) in the guiding layer of the waveguide are excited by adjusting the incident angle of light to fulfill the phase-match condition [20]. Second, when cells are injected into the channel, the intensity of the fluorescence is significantly changed and monitored. Fluorescent faculae are found in the coupled area of the light spot, and the fluorescence intensity of the chromosomes can be measured at a fixed angle [21–23]. Finally, the leaked fluorescence of the single cell through the coupling layer is collected and recorded. The fluorescence intensity is proportional to the number of chromosomes. By analyzing the intensity of the leaked fluorescence, we can draw a map to distinguish the number of the chromosomes from the single cell and detect whether it is normal or not. In this way, we precisely and promptly detect the reproductive cells of male mice, lung cancer cells, and surviving cancer cells after treatment of cancer by the three categories of therapy.

In a previous study [24,25], the low intensity of the fluorescence can be detected by our strong coupling waveguide. Because the different fluorescent intensities correspond to different chromosome numbers, it is possible to distinguish abnormalities by a cell’s chromosome number. Thus, we used nonsmall cell lung cancer (NSCLC) cells from a 53-year-old male whose cancer had progressed on multiple prior lines of chemotherapy and epidermal growth factor receptor tyrosine kinase inhibitors; resistance biopsy was T790M+ without evidence of the C797S mutation. The biopsies were separated and processed as cell solutions. Figure 1(a) displays the section of NSCLC cells under a microscope. Different cancer cell lines with DAPI stained chromosomes are shown in Figs. 1(b)–1(e) under a 60-fold microscope. Figure 1(f) illustrates a brief flow diagram of the separation of different cells. The separations of cells stained by DAPI are based on their sizes. The cells are divided into three groups based on their number of chromosomes. A normal group indicates two sets of chromosomes (all human beings have 46 chromosomes in 23 matching pairs [26]), whereas the abnormal group contains 23, 69, or many more chromosomes. Figures 1(g)–1(i) show the normal microscopy images, abnormal microscopy images, and chromosome fluorescent intensity, respectively.

Based on the method described above, we succeed in discriminating the normal and lung cancer cells according to the intensity of the chromosome fluorescence [Fig. 1(j)] in the map. In the middle of the chart, there is a normal zone between the red-dotted lines: the top and bottom red-dotted lines represent the intensity of the chromosome fluorescence to be 1000 and 750, respectively. If the intensity of the measured fluorescence lies beyond this region, then the cell is likely to be abnormal. An abnormal chromosome number may suggest that the cell is cancerous.

In order to prove that our detection method can distinguish the number of chromosomes in a single cell, we use reproductive cells of male mice which have various chromosome numbers due to abnormal spermiogenesis. The reproductive cells with a normal or an abnormal number of chromosomes come from our research partner (Shanghai Key Lab of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University) [27]. As shown in Figs. 2(a)–2(c), we obtain haploid, diploid, and tetraploid sperm cells. Flow cytometry evaluates the separation of different testicular reproductive cells. The separations of cells are based on their sizes. The reproductive cells (haploid, diploid, and tetraploid sperm cells) are prepared separately in a suitable concentration (1−102μL−1) and injected into the sample cavity of the waveguide. Samples are washed with cold PBS three times and mixed with 3.7% formaldehyde at room temperature (RT) for 20 min followed by rinsing with phosphate buffer solution. Cells are permeabilized with PBS containing 0.5% Triton X-100 for 8 min. Then, we show the enhancement of the fluorescence from the chromosome in our waveguide, as shown in Figs. 2(d)–2(j).

Figures 2(d)–2(f) show the fluorescence microscope ×103 imaging of different germ cells. The chromosomes are dyed by DAPI in the cells. Figures 2(g)–2(i) show the intensity of the fluorescence at the coupling angles when single haploid, diploid, and tetraploid sperm cells are injected into the waveguide, respectively. The central fluorescent wavelength is always 540 nm, and the value of the fluorescent peak is 923, 2005, and 3680 for a single haploid, diploid, and tetraploid sperm cell, respectively [Fig. 2(j)]. Obviously, the fluorescent intensities can distinguish the number of chromosomes in a single cell.

In the validation above, we find that the method based on our strong coupling waveguide can detect chromosomal abnormalities. Moreover, it can be further used to precisely monitor the process of cancer therapy. As shown in Fig. 3, the in vitro results demonstrate that our optical fluorescence detection map exhibits a strong and specific ability to distinguish cancer and normal cells via chromosome fluorescence. It is necessary to employ this method for in vivo studies of cancer therapy. In this experiment, 4T1 tumor-bearing mice (Laboratory of Regeneromics, School of Pharmacy, Shanghai Jiao Tong University) are used. Serial coronal PET/CT (positron emission tomography/computed tomography) images of the 4T1 tumor-bearing mice at different time points of post-injection of the targeted drug are shown in Figs. 3(a) and 3(b). Tumors are indicated by the yellow arrowheads. After the targeted drug in cancer therapy [Fig. 3(a)], the tumor can no longer be seen via PET/CT imaging as shown in Fig. 3(b).

Meanwhile, we detect the cells from mice after therapy via our waveguide. The intensity of the chromosome fluorescence after therapy is shown in Fig. 3(c). The fluorescent data show some cells are in the abnormal zone (the top and bottom dotted lines above 1100 or below 500). Then, a proportion of these mice after therapy labeled as Group 1 are bred in highly controlled research environments for 1 week or more. The PET/CT images after 1 week [Fig. 3(d)] demonstrate that tumors recur. And another part of these mice labeled as Group 2 are with continued drug under continuous monitoring until the data of the chromosome fluorescence intensity [Fig. 3(e)] are between the dotted lines (normal region), which represents tumors are completely killed in our fluorescence detection map. Observation is continued for 1 and 2 weeks after treatment. PET/CT images after 1 and 2 weeks [Fig. 3(f)] show that the tumors disappear in Group 2. In brief, the precise and easy diagnosis of abnormal cells is useful to cancer therapy. The results show that our ultrasensitive detection of an abnormal chromosome number is useful in cancer monitoring and treatment.

This work reports a fluorescence detection map which can detect a single cancer cell in real time via a strong coupling waveguide. In our work, the weak intensity of the fluorescence has been enhanced by the resonance with a UOM in the waveguide, resulting in the chromosome fluorescence of a single live cell being able to be identified without a high-resolution microscope. Based on the above, we propose the method by detecting the NSCLC, which can be applied to monitor the chromosomal abnormalities. Subsequently, the different testicular reproductive cells including haploid, diploid, and tetraploid sperm cells have been distinguished with the map. According to the in vivo experiments in mice, we find that the method can improve the chance of killing any remaining cancer cells to prevent the cancer from recurrence after cancer therapy, facilitating personalized cancer diagnosis and therapy. The single live cell at adjacent points after treatments for cancer can be monitored in real time by the waveguide in order to monitor the metastasis of the tumor in time. Moreover, for distant points the waveguide can be used as an adjuvant device to the cancer diagnosis, which possesses sensitive detection ability to compensate the shortcoming of existing medical conditions. Early and precise cancer diagnosis can substantially improve patient surviving due to our real-time ultrasensitive detection. The researchers, who have been working diligently to find a personalized cure for cancer, can get a useful and new way to monitor and treat cancers.

Authors claim: all applicable institutional and national guidelines for the care and use of animals were followed.

The optical setup is illustrated in Fig. 4(a). The system is relatively simple due to the free space coupling technique. A computer-controlled θ/2θ goniometer is applied for the accurate angular scanning of the incident light to ensure efficient energy coupling. The size of the rectangular sample channel is 10mm×4mm×0.4mm. All of the parts are optically contacted with excellent parallelism. A CCD (uEye Cockpit) is used to record signal data collected by closeup lenses perpendicular to the reflected light. In our approach, a photodiode monitors the variation of the reflected light intensity. The details are presented in Figs. 4(b) and 4(c). Figure 4(b) shows the diagram of the microsyringe. And the structure of the metal-cladding optofluidic waveguide is indicated in Fig. 4(c). Figures 4(d) and 4(e) show the details about attenuating the total reflection absorption peak (ATR). In experiments, the flow velocity is 10 μL/s, and the time interval of the recorded data is Δt=1min. After passing through a collimating system, a collimated beam with a 473 nm (CW) and 15 mW laser is coupled to the metal-cladding layer of the waveguide which rotates with the goniometer to excite the UOMs. After reaching the coupling angle θi, more than 99% incident light will be coupled into the waveguide. Therein, the effective index Neff of the waveguide is between 0 and 1 due to the symmetrical metal cladding, the result of which is that the light can still be trapped in the waveguide layer without leakage. The liquid with a low refractive index is injected into the waveguide interlayer, so that the interaction between light and matter can be fully achieved in the waveguide. Meanwhile, the stimulation results show a molecular structure or weight change in the sample, corresponding to a change of refractive index ∼10−6, which will consequently generate a 0.012° angle shift as shown in Fig. 4(e).

As shown in Fig. 2(j), the different number of chromosomes can be detected by the intensity of the fluorescence change in the waveguide with a sperm cell suspension. But, if the suspension is mixed with impurities, just the detection of the refractive index may fail to distinguish the number of the chromosomes in the cell. Therefore, a method to achieve specific signals from the chromosomes in the waveguide is needed to distinguish a single normal cell and an abnormal one. The fluorescence of the chromosome is a specific signal, and the intensity of the fluorescence is related to the number of chromosomes.

As shown in Fig. 5, we detect the intensity of the fluorescence at coupling angles θi=0.41°, 0.43°, and 0.45°, when the haploid, diploid, and tetraploid sperm cells are injected into the waveguide, respectively. As indicated in Fig. 5(a), the incident light knocks at the top of the waveguide at the coupling angle, and there is light power that was coupled into the waveguide. Thus, black lines are present on the reflected spots captured by the CCD. More detailed information about the incident light and reflected light is provided in Fig. 5(b). The fluorescent intensity remains stable at θi=0.41°, 0.43°, and 0.45° [Fig. 5(c)]. Furthermore, the haploid, diploid, and tetraploid sperm cells can still be distinguished via detection of the fluorescent intensity.

The mouse sperm used was previously described [28,29]. Briefly, KunMing mice (Shanghai Laboratory Animal Center of the Chinese Academy of Sciences) were used in this study. Animal experiments were performed according to the institutional guidelines for ethics (Approval number 86/609/EEC-24/11/86). All of the experimental procedures were approved by the Animal Ethics Committee of Shanghai Jiao Tong University (No. IACUC-2018-010). The housing facility is a barrier housing facility, and it is maintained according to the national standards contained in “Laboratory Animal-Requirements of Environment and Housing Facilities” (GB 14925-2001). The care of laboratory animals and the animal experimental operation conformed to the “Beijing Administration Rule of Laboratory Animals.”

We used 10 cages (at least) of newborn male mice in each experiment, and the number of newborn male mice was more than 50. Newborn male mice were randomly divided into two groups and were caged separately. Ear cropping was used to identify the Pd-treated group. PbCl2 (1.5 mg/kg BW [30,31]) was administered in the treated groups via intraperitoneal injection once every 2 days from postnatal days 10 to 60. The control groups were injected with the same volume of normal saline. All of the experiments were repeated three times. Based on a previous study of reproductive system development [32], we selected postnatal days 20, 40, and 60 to indicate the different developmental stages. The testes from the mice in each group were dissected and placed in 1 mL PBS to release the reproductive cells, and the tissues were removed and washed with PBS after 15 min.

To generate the 4T1 tumor model, 4–5-week-old female Qui Gue TM-PDX mice were purchased from JNO (Jennio-bio, Guangzhou, China). Tumors were established by subcutaneously injecting 2×106 cells which were suspended in 100 μL of 1:1 mixture of RPMI 1640 and matrigel (BD Biosciences, Franklin Lake, NJ) into the front flank of mice. The tumor sizes were monitored every other day by CT, and the animals were subjected to in vivo experiments when the tumor diameter was over 5 mm.

PET and PET/CT scans at various time points were done post-injection using a microPET/microCT Inveon rodent model scanner (Siemens Medical Solution USA, Inc.) with 2 μm resolution. The scan space was 80mm×200mm. Moreover, the image reconstruction and the ROI analysis of the PET data were presented as a percentage injected dose per gram of tissue (% ID/g). Tumor-bearing mice were each injected with 5–10 MBq of contrast agent via the tail vein before serial PET scans.

Frozen tissue slices (7 μm thickness) were fixed with cold acetone and stained for endothelial marker CD31 through the use of a rat antimouse CD31 antibody and a Cy3-labeled donkey antirat lgG. The tissue slice was also incubated with 2 μg/mL of acridine-orange-labeled goat antihuman lgG for visualization of the chromosome. All of the images were acquired with a DYF-880 fluorescent microscope.

The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL). The cells were incubated in a humidified atmosphere with 5% CO2 at 37°C.

Acridine orange and Cy3-labeled secondary antibodies were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Absolute ethanol, sodium chloride (NaCl), and doxorubicin hydrochloride (DOX) were purchased from Fisher Scientific. Water and all buffers were of Millipore grade and pretreated with a Chelex 100 resin to ensure that the aqueous solution was free of heavy metals. All of the chemicals were used as received without further purification.

Acknowledgment. The authors acknowledge support from the State Key Laboratory of Advanced Optical Communication Systems and Networks, and also thank the Ruijin Hospital, Shanghai Jiao Tong University School of Medicine.