Microfluidic based single cell or droplet manipulation: Methods and applications.

The isolation of single cell or droplet is first and crucial step to single-cell analysis, which is important for cancer research and diagnostic methods. This review provides an overview of technologies that are currently used or in development to realize the isolation. Microfluidic based manipulation is an emerging technology with the distinct advantages of miniaturization and low cost. Therefore, recent developments in microfluidic isolated methods have attracted extensive attention. We introduced herein five strategies based on microfluid: trap, microfluidic discrete manipulation, bioprinter, capillary and inertial force. For every technology, their basic principles and features were discussed firstly. Then some modified approaches and applications were listed as the extension. Finally, we compared the advantages and drawbacks of these methods, and analyzed the trend of the manipulation based on microfluidics.

Droplet magnetic-controlled microfluidic chip integrated nucleic acid extraction and amplification for the detection of pathogens and tumor mutation sites.

Traditional nucleic acid extraction and detection is based on open operation, which may cause cross-contamination and aerosol formation. This study developed a droplet magnetic-controlled microfluidic chip integrated nucleic acid extraction, purification and amplification. The reagent is sealed in oil to form a droplet, and the nucleic acid is extracted and purified by controlling the movement of the magnetic beads (MBs) through a permanent magnet, ensuring a closed environment. This chip can automatically extract nucleic acid from multiple samples within 20 min, and can be directly placed in the in situ amplification instrument for amplification without further transfer of nucleic acid, characterized by simple, fast, time-saving and labor-saving. The results showed that the chip was able to detect <10 copies/test SARS-CoV-2 RNA, and EGFR exon 21 L858R mutations were detected in H1975 cells as low as 4 cells. In addition, on the basis of the droplet magnetic-controlled microfluidic chip, we further developed a multi-target detection chip, which used MBs to divide the nucleic acid of the sample into three parts. And the macrolides resistance mutations A2063G and A2064G, and the P1 gene of mycoplasma pneumoniae (MP) were successfully detected in clinical samples by the multi-target detection chip, providing the possibility for future application in the detection of multiple pathogens.

A microfluidic immunosensor for automatic detection of carcinoembryonic antigen based on immunomagnetic separation and droplet arrays.

Diagnosis of cancer by biomarkers plays an important role in human health and life. However, current laboratory techniques for detecting cancer biomarkers still require laborious and time-consuming operation by skilled operators and associated laboratory instruments. This work presents a colorimetric biosensor for the rapid and sensitive detection of carcinoembryonic antigen (CEA) based on an automated immunomagnetic separation platform and a droplet array microfluidic chip with the aid of an image analysis system. Immunomagnetic nanoparticles (MNPs) were used to capture CEA in the samples. CEA-detecting antibodies and horseradish peroxidase (HRP) were modified on polystyrene microspheres (PS), catalysing hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine (TMB) as signal outputs. Color reaction data were analyzed to establish a CEA concentration standard curve. The movement of MNPs between droplets in the microfluidic chip is achieved using an automatically programmable magnetic control system. This colorimetric biosensor has been used for the simultaneous detection of six CEA samples ranging from 100 pg mL-1 to 100 ng mL-1 with a detection limit of 14.347 pg mL-1 in 10 min, following the linear equation: y = -4.773 ln(x) + 156.26 with a correlation of R2 = 0.9924, and the entire workflow can be completed within 80 minutes. The microfluidic immunosensor designed in this paper has the advantages of low cost, automation, low sample consumption, high throughput, and promising applications in biochemistry.

Detection of EGFR gene with a droplet digital PCR chip integrating a double-layer glass reservoir.

The lack of reliable and practical method for detecting rare hot mutation of epidermal growth factor receptor (EGFR) in circulating tumor DNA (ctDNA) for lung cancer has remained a challenge for general clinical application due to excess wild type DNA in clinical samples. In this study, we developed a droplet digital PCR (ddPCR) platform, integrating a PDMS chip and double-layer glass reservoir. The duplex T-junction droplet generators in PDMS chip can produce about one million uniform droplets of 4.187 pL within ∼10 min, which were then stored in the glass reservoir. The double-layer glass reservoir can protect droplets from evaporation and breaking, solving the problem of instability during thermal-cycling. The quantitative capabilities of the ddPCR chip were evaluated by testing EGFR exon gene 21, with a good linear correlation in the wide range of 101 to 106 copies/µL (R2 = 0.9998). We then demonstrated that the proposed ddPCR device can recognize rare EGFR L858R mutation under a background of 106 copies/µL wild-type DNA at a sensitivity of 0.0001%. Finally, we demonstrated this ddPCR platform could identify low amount of EGFR L858R mutation in ctDNA and CTCs of patients with lung cancer.

The Discovery of Novel Circulating Cancer-Related Cells in Circulation Poses New Challenges to Microfluidic Devices for Enrichment and Detection.

Circulating tumor cells (CTCs) enumeration has been widely used as a surrogate predictive marker for early diagnoses, the evaluation of chemotherapy efficacy, and cancer prognosis. Microfluidic technologies for CTCs enrichment and detection have been developed and commercialized as automation platforms. Currently, in addition to CTCs, some new types of circulating cancer-related cells (e.g., CCSCs, CTECs, CAMLs, and heterotypic CTC clusters) in circulation are also reported to be correlated to cancer diagnosis, metastasis, or prognosis. And they widely differ from the conventional CTCs in positive markers, cellular morphology, or size, which presents a new technological challenge to microfluidic devices that use affinity-based capture methods or size-based filtration methods for CTCs detection. This review focuses on the biological and physical properties as well as clinical significance of the novel circulating cancer-related cells, and discusses the challenges of their discovery to microfluidic chip for enrichment. Finally, the current challenges of CTCs detection in clinical application and future opportunities are also discussed.

Wet-Etched Microchamber Array Digital PCR Chip for SARS-CoV-2 Virus and Ultra-Early Stage Lung Cancer Quantitative Detection.

We report a novel design of chamber-based digital polymerase chain reaction (cdPCR) chip structure. Using a wet etching process and silicon-glass bonding, the chamber size can be adjusted independently of the process and more feasibly in a normal lab. In addition, the structure of the chip is optimized through hydrodynamic computer simulations to eliminate dead space when the sample is injected into the chip. The samples will be distributed to each separated microchambers for an isolated reaction based on Poisson distribution. Due to the difference in expansion coefficients, isolation of the sample in the microchambers by the oil phase on top ensures homogeneity and independence of the sample in the microchambers. The prepared microarray cdPCR chip enables high-throughput and high-sensitivity quantitative measurement of the SARS-CoV-2 virus gene and the mutant lung cancer gene. We applied the chip for the detection of different concentrations of the mix containing the open reading frame 1ab (ORF1ab) gene, the most specific and conservative gene region of the SARS-CoV-2 virus. In addition to this, we also successfully detected the fluorescence of the epidermal growth factor receptor (EGFR) mutant gene in independent microchambers. At a throughput of 46 200 microchambers, solution mixtures containing both genes were successfully tested quantitatively, with a detection limit of 10 copies/µL. Importantly, the chips are individually inexpensive and easy to industrialize. In addition, the microarray can provide a unified solution for other viral sequences, cancer marker assay development, and point-of-care testing (POCT).

Single-cell genetic analysis of lung tumor cells based on self-driving micro-cavity array chip.

Lung cancer is one of the common malignant tumors with a high incidence and mortality rate. Targeted therapies are efficient on lung cancer patients with specific gene mutations. Circulating tumor cells (CTCs) are used for liquid biopsy, providing genetic information for lung cancer treatment selection and prognosis. We developed a less costly self-driving micro-cavity array for simple molecular analysis at a single cell level to examine the genetic make-up of CTCs. This chip integrated sample detection structure and vacuum driving system to achieve cell loading, lysing, isothermal amplification (LAMP), and signal read-out on one chip. We used the "film-polydimethylsiloxane (PDMS) chip-film" structure and oil sealing method during amplification reaction to minimize water loss. We then conducted a LAMP assay using the self-driving device to detect epidermal growth factor receptor (EGFR) L858R mutation and identified an excellent linear in the range between 101-104 copies/µL (R2 = 0.997). We finally assessed the EGFR L858R gene expression of lung tumor cells (H1975 cells) as putative CTCs using the proposed detection platform. We discovered its ability to perform genetic analysis at the single-cell level. The EGFR L858R mutational gene expression levels were different in H1975 cells. In conclusion, the self-driving micro-cavity array is a less costly and simple tool for mutational gene profiling of single lung CTC. Besides, it can be used in personalized therapy and efficacy monitoring.

EGFR mutation detection of lung circulating tumor cells using a multifunctional microfluidic chip.

Microfluidics has become a reliable platform for circulating tumor cells (CTCs) detection because of its high integration, small size, low consumption of reagents and rapid response. Here, we developed a multifunctional microfluidic device consists of three parts, including CTCs capture area, single-layer membrane valves area, and microcavity nucleic acid detection and analysis region based on digital polymerase chain reaction (dPCR), allowing CTCs capture, lysis, and genetic characterization to be performed on a single chip. The CTCs capture chip is coupled to the nucleic acid detection chip via a control valve. CTCs were firstly trapped in the CTC capture area, and then lysed using proteinase K to release nucleic acids. Subsequently CTCs lysate was transferred into nucleic acid detection area consisting of 12800 micro-cavity chambers for nucleic acids detection. To evaluate the performance of this chip, this study detected EGFR-L858R mutation in lung cancer cell lines H1975 and A549 cells, as well as leukocytes from normal donors. The results showed that positive signals were only observed in H1975 cells, and the detected value had a high linear relationship with the expected value (R2 = 0.9897). In conclusion, this multi-functional microfluidic chip that integrates CTCs capture, lysis and nucleic acid detection can successfully detect gene mutations in CTCs, providing reference for tumor-targeted drugs and precise diagnosis and treatment.

Self-Assembled Hybrid Nanogel as a Multifunctional Theranostic Probe for Enzyme-Regulated Ultrasound Imaging and Tumor Therapy.

Multifunctional theranostic nanoprobes integrated with stimuli-responsive imaging and therapeutic capabilities have shown great potential to enhance the early cancer diagnostic efficacy and therapeutic efficiency. Elevated levels of lactate and hydrogen peroxide have been considered as the characteristic feature of the tumor microenvironment and can thus be exploited for developing promising theranostic strategies. We demonstrate here that the biocompatible and responsive enzyme-based nanogel probe has been designed as a promising theranostic tool to target high lactate and hydrogen peroxide for ultrasound imaging (US) and cancer treatment. We encapsulate the dual enzyme lactate oxidase (LOD) and catalase (CAT) into the self-assembled nanogels to fabricate responsive nanoprobe LOD/CAT-loaded nanogels (LCNGs). The nanoprobes can respond to the lactate and H2O2 rich tumor microenvironment to generate abundant oxygen, which further accumulates into microbubbles for enhanced US imaging. Besides, LCNGs@DOX has been further created by integrating the nanoprobes with doxorubicin (DOX) for cancer therapy. Both in vitro and in vivo results demonstrate enhanced US imaging and effective cell proliferation inhibition of LCNGs@DOX, allowing the preparation of safe and efficient theranostic nanoprobes capable of responsive US imaging and treating tumors.

A label-free microfluidic chip for the highly selective isolation of single and cluster CTCs from breast cancer patients.

BACKGROUND: Circulating tumor cells (CTCs) existing in peripheral blood can be used to predict the prognosis and survival of cancer patients. The study was designed to detect circulating tumor cells and circulating tumor single cell genes by applying microfluidic chip technology. It was used to explore the clinical application value in breast cancer. METHODS: We have developed a size-based CTCs sorting microfluidic chip, which contains a hexagonal array and a micro-pipe channel array to isolate and confirm both single CTCs and CTCs clusters. The sorting performance of the as-fabricated chip was tested by analyzing the clinical samples collected from 129 breast cancer patients and 50 healthy persons. RESULTS: In this study, the chip can detect different immunophenotypes of CTCs in breast cancer patients. It was found that the new microfluidic device had high sensitivity (73.6%) and specificity (82.0%) in detecting CTCs. By detecting the blood samples of 129 breast cancer patients and 50 healthy blood donors, it was found that the number of CTCs was not associated with clinical factors such as age, gender, pathological type, and tumor size of breast cancer patients (P > 0.05), but was associated with TNM staging of breast cancer, with or without metastasis (P < 0.005). There was a statistically significant difference in the number of CTCs between luminal A (ER+/PR+/HER2-) and HER-2+ (ER-/PR-/HER2+) (P < 0.05). The best cut-off level distinguished by CTC between the breast cancer patients and the healthy persons was 3.5 cells/mL, with 0.845 for AUC-ROC, 0.790-0.901 for 95% CI, 73.6% for sensitivity, and 82% for specificity (P = 0.000). The combination of CTC, CEA, CA125 and CA153 can provide more effective breast cancer screening. CONCLUSIONS: The CTCs analysis method presented here doesn't rely on the specific antibody, such as anti-EpCAM, which would avoid the missed inspection caused by antibody-relied methods and offer more comprehensive biological information for clinical breast cancer diagnosis and treatment.

A centrifugal microfluidic emulsifier integrated with oil storage structures for robust digital LAMP.

Centrifugal droplet-based microfluidic devices have been applied to biomedical analysis and diagnostics recently. However, in centrifugal droplet-based microfluidic devices, droplets are tightly packed (i.e., the oil film between neighbouring droplets is thin). Therefore, droplet coalescence usually occurs especially during thermal incubation process. To preserve individual droplets in the devices, we report a new design for monodisperse droplet generation and storage that exploits a centrifugal configuration for droplet emulsification and oil-storage structures (OSSs) for regulation of the thickness of oil film between neighbouring droplets. The centrifugal emulsifier was well designed to ensure uniform droplet generation. Meanwhile, the OSSs could store oil during centrifugal emulsification while release oil before thermal incubation, which "loosen" tightly packed droplets to prevent droplets from coalescing. In this paper, the working process of OSS was analysed, and its shape and size were optimized. Then, the optimized OSSs were integrated into a centrifugal emulsifier for droplet digital loop mediated isothermal amplification (ddLAMP) by which detection of JAK2 V617F mutation within myeloproliferative neoplasms with a dynamic range of 101 to 104 copies per µL was achieved. We anticipate that the simplicity and robustness of our system make it attractive as an inexpensive and easy-to-operate device for DNA amplification, particularly applicable in point-of-care settings.

EGFR point mutation detection of single circulating tumor cells for lung cancer using a micro-well array.

In view of their critical function in metastasis, characterization of single circulating tumor cells (CTCs) can provide important clinical information to monitor tumor progression and guide personal therapy. Single-cell genetic analysis methods based on microfluidics have some inherent shortcomings such as complicated operation, low throughput, and expensive equipment requirements. To overcome these barriers, we developed a simple and open micro-well array containing 26,208 units for either nuclear acids or single-cell genetic analysis. Through modification of the polydimethylsiloxane surface and optimization of chip packaging, we addressed protein adsorption and solution evaporation for PCR amplification on a chip. In the detection of epidermal growth factor receptor (EGFR) exon gene 21, this micro-well array demonstrated good linear correlation at a DNA concentration from 1 × 101 to 1 × 105 copies/µL (R2 = 0.9877). We then successfully integrated cell capture, lysis, PCR amplification, and signal read-out on the micro-well array, enabling the rapid and simple genetic analysis of single cells. This device was used to detect duplex EGFR mutation genes of lung cancer cell lines (H1975 and A549 cells) and normal leukocytes, demonstrating the ability to perform high-throughput, massively parallel duplex gene analysis at the single-cell level. Different types of point mutations (EGFR-L858R mutation or EGFR-T790M mutation) were detected in single H1975 cells, further validating the significance of single-cell level gene detection. In addition, this method showed a good performance in the heterogeneity detection of individual CTCs from lung cancer patients, required for micro-invasive cancer monitoring and treatment selection.

Highly sensitive detection and mutational analysis of lung cancer circulating tumor cells using integrated combined immunomagnetic beads with a droplet digital PCR chip.

Circulating tumor cells (CTCs) have become an important biomarker for liquid biopsy to monitor tumor progression and indicate response to therapies. Many epithelial cellular adhesion molecule (EpCAM) dependent CTC isolation methods have been developed, which have a limitation for low EpCAM expressed tumor cells. In an effort to overcome these drawbacks, we developed combined immunomagnetic beads (EpCAM, Mucin1 and epidermal growth factor receptor) to sensitively isolate CTCs for immunofluorescence analysis and genetic characterization. With this combined immunomagnetic beads, 93.35% H446 cells from spiked blood sample can be recovered. We were able to detect CTCs in 127 among 143 patients included in the study (88.8%). Some CTC clusters were captured with the combined magnetic beads system. In 17 of them, CTCs after chemotherapy significantly decreased compared to that before chemotherapy (4.42 (±â€¯3.94) vs. 12 (±â€¯7)/mL, P = 0.002). For subsequent genetic characterization of CTCs, 2 of 6 samples, using a droplet digital PCR (ddPCR) chip, have detectable EGFR L858R mutation in the cells enriched with the combined immunomagnetic beads. In conclusion, this method integrating the combined immunomagnetic beads and the ddPCR chip for CTCs detection can be of potential application in terms of diagnosis, therapeutic evaluation and personalized medicine in lung cancer.

Clinical significance of circulating tumor cells from lung cancer patients using microfluidic chip.

Circulating tumor cells (CTCs) exist in the peripheral blood and have an important role in the disease development, tumor metastasis and clinical surveillance, especially in the process of metastasis. However, the technology of detecting CTCs still had a large challenge since they were rare in the peripheral blood. Here, we developed a size-based microfluidic chip, which contained array and filter channel array that could enrich CTCs from blood samples more quickly and conveniently. Combined with clinical specimen, we analyzed CTCs in 200 lung cancer patients by this microfluidic chip. The microfluidic device has high specificity and sensitivity in detecting CTCs (86.0% sensitivity and 98% specificity). Furthermore, the number of CTCs showed a increasing trend according to the stage of the disease (the mean number of I stage 5.0 ± 5.121 versus II stage 8.731 ± 6.36 versus III stage 16.81 ± 9.556 versus IV stage 28.72 ± 17.39 cells/mL, P < 0.05). The number of CTCs was concurrent with the condition of pathological type and metastasis patients. Compared to conventional markers like CEA, CY211, SCC, CTCs showed a higher positive rate in diagnosed patients. The advanced microfluidic device could capture tumor cells without reliance on cell surface expression markers and provide a fast, convenient, economical method in detecting CTCs, thereby offering potential to design effective and individualized cancer therapies.

Highly sensitive detection of multiple tumor markers for lung cancer using gold nanoparticle probes and microarrays.

Assay of multiple serum tumor markers such as carcinoembryonic antigen (CEA), cytokeratin 19 fragment antigen (CYFRA21-1), and neuron specific enolase (NSE), is important for the early diagnosis of lung cancer. Dickkopf-1 (DKK1), a novel serological and histochemical biomarker, was recently reported to be preferentially expressed in lung cancer. Four target proteins were sandwiched by capture antibodies attached to microarrays and detection antibodies carried on modified gold nanoparticles. Optical signals generated by the sandwich structures were amplified by gold deposition with HAuCl4 and H2O2, and were observable by microscopy or the naked eye. The four tumor markers were subsequently measured in 106 lung cancer patients and 42 healthy persons. The assay was capable of detecting multiple biomarkers in serum sample at concentration of <1 ng mL-1 in 1 h. Combined detection of the four tumor markers highly improved the sensitivity (to 87.74%) for diagnosis of lung cancer compared with sensitivity of single markers. A rapid, highly sensitive co-detection method for multiple biomarkers based on gold nanoparticles and microarrays was developed. In clinical use, it would be expected to improve the early diagnosis of lung cancer.

Analysis of circulating tumor cells from lung cancer patients with multiple biomarkers using high-performance size-based microfluidic chip.

Circulating tumor cells (CTCs) have attracted pretty much attention from scientists because of their important relationship with the process of metastasis. Here, we developed a size-based microfluidic chip containing triangular pillar array and filter channel array for detecting single CTCs and CTC clusters independent of tumor-specific markers. The cell populations in chip were characterized by immune-fluorescent staining combining an epithelial marker and a mesenchymal marker. We largely decreased the whole time of detection process to nearly 1.5h with this microfluidic device. The CTCs were subsequently measured in 77 patients with lung cancer and 39 healthy persons. The microfluidic device allowed for the detection of CTCs with apparent high sensitivity and specificity (82.7% sensitivity and 100% specificity). Furthermore, the total CTC counts were found to be elevated in advanced patients with metastases when compared with those without (20.89±14.57 vs 8.428±5.858 cells/mL blood; P<0.01). Combined epithelial marker and mesenchymal marker analysis of CTCs could provide more information about metastasis in patients than only usage of epithelial marker. In conclusion, the development of the size-based microfluidic device for efficient capture of CTCs will enable detailed characterization of their biological properties and values in cancer diagnosis.

Label-free graphene biosensor targeting cancer molecules based on non-covalent modification.

A label-free immunosensor based on antibody-modified graphene field effect transistor (GFET) was presented. Antibodies targeting carcinoembryonic antigen (Anti-CEA) were immobilized to the graphene surface via non-covalent modification. The bifunctional molecule, 1-pyrenebutanoic acid succinimidyl ester, which is composed of a pyrene and a reactive succinimide ester group, interacts with graphene non-covalently via π-stacking. The succinimide ester group reacts with the amine group to initiate antibody surface immobilization, which was confirmed by X-ray Photoelectron Spectroscopy, Atomic Force Microscopy and Electrochemical Impedance Spectroscopy. The resulting anti-CEA modified GFET sufficiently monitored the reaction between CEA protein and anti-CEA in real-time with high specificity, which revealed selective electrical detection of CEA with a limit of detection (LOD) of less than 100pg/ml. The dissociation constant between CEA protein and anti-CEA was estimated to be 6.35×10-11M, indicating the high affinity and sensitivity of anti-CEA-GFET. Taken together, the graphene biosensors provide an effective tool for clinical application and point-of-care medical diagnostics.

Highly sensitive and selective lateral flow immunoassay based on magnetic nanoparticles for quantitative detection of carcinoembryonic antigen.

Carcinoembryonic antigen (CEA) is an important biomarker in cancer diagnosis. Here, we present an efficient, selective lateral-flow immunoassay (LFIA) based on magnetic nanoparticles (MNPs) for in situ sensitive and accurate point-of-care detection of CEA. Signal amplification mechanism involved linking of detection MNPs with signal MNPs through biotin-modified single-stranded DNA (ssDNA) and streptavidin. To verify the effectiveness of this modified LFIA system, the sensitivity and specificity were evaluated. Sensitivity evaluation showed a broad detection range of 0.25-1000ng/ml for CEA protein by the modified LFIA, and the limit of detection (LOD) of the modified LFIA was 0.25ng/ml, thus producing significant increase in detection threshold compared with the traditional LFIA. The modified LFIA could selectively recognize CEA in presence of several interfering proteins. In addition, this newly developed assay was applied for quantitative detection of CEA in human serum specimens collected from 10 randomly selected patients. The modified LFIA system detected minimum 0.27ng/ml of CEA concentration in serum samples. The results were consistent with the clinical data obtained using commercial electrochemiluminescence immunoassay (ECLIA) (p<0.01). In conclusion, the MNPs based LFIA system not only demonstrated enhanced signal to noise ratio, it also detected CEA with higher sensitivity and selectivity, and thus has great potential to be commercially applied as a sensitive tumor marker filtration system.

Multiplexed detection of lung cancer biomarkers based on quantum dots and microbeads.

We have developed a multiplexed fluoroimmunoassay of three lung cancer biomarkers based on multicolor quantum dots (QDs) as detection elements and micro-magnetic beads as immune carriers. QDs have the ability to simplify multiplexed analysis. In our method, the fluorescent signals derived from three cross-talk-free QD conjugated probes with emission maxima at 525, 585 and 625nm could be analyzed to determine the concentrations of the target proteins. With this system, fragments of cytokeratin 19 (CYRFA 21-1), carcinoembryonic antigen (CEA), and neuron-specific enolase (NSE), were simultaneously detected in a single sample with a low detection limit down to the 1.0ng/mL level (364pg/mL for CYRFA 21-1, 38pg/mL for CEA, 370pg/mL for NSE in a single detection). Additional advantages of the presented method include ease of operation, low cost, and a very low sample volume (20µL).

Bead-based microarray immunoassay for lung cancer biomarkers using quantum dots as labels.

In this study, we developed a multiplex immunoassay system that combines the suspension and planar microarray formats within a single layer of polydimethylsiloxane (PDMS) using soft lithography technology. The suspension format was based on the target proteins forming a sandwich structure between the magnetic beads and the quantum dot (QD) probes through specific antibody-antigen interactions. The planar microarray format was produced by fabricating an array of micro-wells in PDMS. Each micro-well was designed to trap a single microbead and eventually generated a microbead array within the PDMS chamber. The resultant bead-based on-chip assay could be used for simultaneously detecting three lung cancer biomarkers-carcinoembryonic antigen (CEA), fragments of cytokeratin 19 (CYFRA21-1) and neuron-specific enolase (NSE)-in 10 µl of human serum, with a wide linear dynamic range (1.03-111 ng/mL for CEA and CYFRA21-1; 9.26-1000 ng/ml for NSE) and a low detection limit (CEA: 0.19 ng/ml; CYFRA21-1: 0.97 ng/ml; NSE: 0.37 ng/ml; S/N=3). Our micro-well chip does not require complex e-beam lithography or the reactive ion etching process as with existing micro-well systems, which rely on expensive focused ion beam (FIB) milling or optical fiber bundles. Furthermore, the current approach is easy to operate without extra driving equipment such as pumps, and can make parallel detection for multiplexing with rapid binding kinetics, small reagent consumption and low cost. This work has demonstrated the importance of the successful application of on-chip multiplexing sandwich assays for the detection of biomarker proteins.