Effect of Cyclin-Dependent Kinase 4/6 Inhibitors on Circulating Cells in Patients with Metastatic Breast Cancer.

The combination of cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) with endocrine therapy (ET) is the standard-of-care for estrogen receptor (ER)-positive, HER2-negative (ER+/HER2- advanced/metastatic breast cancer (mBC). However, the impact of CDK4/6i on circulating immune cells and circulating tumor cells (CTCs) in patients receiving CDK4/6i and ET (CDK4/6i+ET) remains poorly understood. This was a prospective cohort study including 44 patients with ER+/HER2- mBC treated with CDK4/6i+ET in either first or second line. Peripheral blood samples were collected before (baseline) and 3 months (t2) after therapy. Immune cell's subsets were quantified by flow cytometry, and microfluidic-captured CTCs were counted and classified according to the expression of cytokeratin and/or vimentin. Patients were categorized according to response as responders (progression-free survival [PFS] ≥ 6.0 months; 79.1%) and non-responders (PFS < 6.0 months; 20.9%). CDK4/6i+ET resulted in significant changes in the hematological parameters, including decreased hemoglobin levels and increased mean corpuscular volume, as well as reductions in neutrophil, eosinophil, and basophil counts. Specific immune cell subsets, such as early-stage myeloid-derived suppressor cells, central memory CD4+ T cells, and Vδ2+ T cells expressing NKG2D, decreased 3 months after CDK4/6i+ET. Additionally, correlations between the presence of CTCs and immune cell populations were observed, highlighting the interplay between immune dysfunction and tumor dissemination. This study provides insights into the immunomodulatory effects of CDK4/6i+ET, underscoring the importance of considering immune dynamics in the management of ER+/HER2- mBC.

Nanofluidic resistive pulse sensing for characterization of extracellular vesicles.

This paper describes the development, design and characterization of a resistive pulse sensing (RPS) system for the analysis of size distributions of extracellular vesicles (EVs). The system is based on microfluidic chips fabricated using soft-lithography and operated in pressure-driven mode. This fabrication approach provided reproducible pore dimensions and the best performing chip design enabled, without calibration, sizing of both 252 nm and 460 nm test particles within 8% of theoretically calculated values, based on the size specifications provided by suppliers. The number concentration measurement had higher variations and without calibration provided estimates within an order of magnitude, for sample concentrations across 4 orders of magnitude. The RPS chips could also measure successfully EVs and other biological nanoparticles in purified samples from cell culture media and human serum. A compact, fast and inexpensive RPS system based on this design could be an attractive alternative to current gold-standard techniques for routine characterization of EV samples.

Isolation of acute myeloid leukemia blasts from blood using a microfluidic device.

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and associated with poor prognosis. Unfortunately, most of the patients that achieve clinical complete remission after the treatment will ultimately relapse due to the persistence of minimal residual disease (MRD), that is not measurable using conventional technologies in the clinic. Microfluidics is a potential tool to improve the diagnosis by providing early detection of MRD. Herein, different designs of microfluidic devices were developed to promote lateral and vertical mixing of cells in microchannels to increase the contact area of the cells of interest with the inner surface of the device. Possible interactions between the cells and the surface were studied using fluid simulations. For the isolation of leukemic blasts, a positive selection strategy was used, targeting the cells of interest using a panel of specific biomarkers expressed in immature and aberrant blasts. Finally, once the optimisation was complete, the best conditions were used to process patient samples for downstream analysis and benchmarking, including phenotypic and genetic characterisation. The potential of these microfluidic devices to isolate and detect AML blasts may be exploited for the monitoring of AML patients at different stages of the disease.

Minimizing false positives for CTC identification.

BACKGROUND: Cancer is a leading cause of death worldwide, with metastasis playing a significant role. Circulating Tumour Cells (CTCs) can provide important real-time insights into tumour heterogeneity and clonal evolution, making them an important tool for early diagnosis and patient monitoring. Isolated CTCs are typically identified by immunocytochemistry using positive biomarkers (cytokeratin) and exclusion biomarkers (CD45). However, some white blood cell (WBC) populations can express low levels of CD45 and stain non-specifically for cytokeratin, increasing their risk of misclassification as CTCs. There is a clear need to improve CTC detection and enumeration criteria to unequivocally eliminate interfering WBC populations. RESULTS: This study showed that, indeed, some granulocyte subpopulations expressed low levels of CD45 and stained non-specifically for cytokeratin, misidentifying them as CTCs. These same cells, however, strongly expressed CD15, allowing them to be identified as WBCs and excluded from CTC classification. Flow cytometry confirmed the specificity of the CD15 antibody for the granulocyte subpopulation. False positives were considerably reduced from 25 % to 0.2 % by double exclusion, combining a CD15 antibody with a highly specific CD45 antibody. Furthermore, complete elimination of potential false positives was achieved using double exclusion in combination with improved selection of cytokeratin antibody. The study emphasises the importance of a robust exclusion criteria and high antibody specificity in CTC immuno-assays for accurate identification of CTC candidates and thorough exclusion of interfering WBC subpopulations. SIGNIFICANCE: This study demonstrated how misidentifying a granulocyte subpopulation can lead to inaccurate CTC evaluation. However, sensitivity and specificity of CTC identification may be improved by using high-performing antibodies and by including a second exclusion biomarker, in turn, allowing for a more comprehensive clinical application of CTCs.

Clinical Validation of a Size-Based Microfluidic Device for Circulating Tumor Cell Isolation and Analysis in Renal Cell Carcinoma.

Renal cell carcinoma (RCC) presents as metastatic disease in one third of cases. Research on circulating tumor cells (CTCs) and liquid biopsies is improving the understanding of RCC biology and metastases formation. However, a standardized, sensitive, specific, and cost-effective CTC detection technique is lacking. The use of platforms solely relying on epithelial markers is inappropriate in RCC due to the frequent epithelial-mesenchymal transition that CTCs undergo. This study aimed to test and clinically validate RUBYchip™, a microfluidic label-free CTC detection platform, in RCC patients. The average CTC capture efficiency of the device was 74.9% in spiking experiments using three different RCC cell lines. Clinical validation was performed in a cohort of 18 patients, eight non-metastatic (M0), five metastatic treatment-naïve (M1TN), and five metastatic progressing-under-treatment (M1TP). An average CTC detection rate of 77.8% was found and the average (range) total CTC count was 6.4 (0-27), 101.8 (0-255), and 3.2 (0-10), and the average mesenchymal CTC count (both single and clustered cells) was zero, 97.6 (0-255), and 0.2 (0-1) for M0, M1TN, and M1TP, respectively. CTC clusters were detected in 25% and 60% of M0 and M1TN patients, respectively. These results show that RUBYchip™ is an effective CTC detection platform in RCC.

Current and Emerging Techniques for Diagnosis and MRD Detection in AML: A Comprehensive Narrative Review.

Acute myeloid leukemia (AML) comprises a group of hematologic neoplasms characterized by abnormal differentiation and proliferation of myeloid progenitor cells. AML is associated with poor outcome due to the lack of efficient therapies and early diagnostic tools. The current gold standard diagnostic tools are based on bone marrow biopsy. These biopsies, apart from being very invasive, painful, and costly, have low sensitivity. Despite the progress uncovering the molecular pathogenesis of AML, the development of novel detection strategies is still poorly explored. This is particularly important for patients that check the criteria for complete remission after treatment, since they can relapse through the persistence of some leukemic stem cells. This condition, recently named as measurable residual disease (MRD), has severe consequences for disease progression. Hence, an early and accurate diagnosis of MRD would allow an appropriate therapy to be tailored, improving a patient's prognosis. Many novel techniques with high potential in disease prevention and early detection are being explored. Among them, microfluidics has flourished in recent years due to its ability at processing complex samples as well as its demonstrated capacity to isolate rare cells from biological fluids. In parallel, surface-enhanced Raman scattering (SERS) spectroscopy has shown outstanding sensitivity and capability for multiplex quantitative detection of disease biomarkers. Together, these technologies can allow early and cost-effective disease detection as well as contribute to monitoring the efficiency of treatments. In this review, we aim to provide a comprehensive overview of AML disease, the conventional techniques currently used for its diagnosis, classification (recently updated in September 2022), and treatment selection, and we also aim to present how novel technologies can be applied to improve the detection and monitoring of MRD.

Advances in Microfluidics for the Implementation of Liquid Biopsy in Clinical Routine.

In recent years, we have seen major advances in the field of liquid biopsy and its implementation in the clinic, mainly driven by breakthrough developments in the area of molecular biology. New developments have seen an integration of microfluidics and also biosensors in liquid biopsy systems, bringing advantages in terms of cost, sensitivity and automation. Without a doubt, the next decade will bring the clinical validation and approval of these combined solutions, which is expected to be crucial for the wide implementation of liquid biopsy systems in clinical routine.

Establishing an Expert Consensus on Key Indicators of the Quality of Life among Breast Cancer Survivors: A Modified Delphi Study.

(1) Background: The needs of cancer survivors are often not reflected in practice. One of the main barriers of the use of patient-reported outcomes is associated with data collection and the interpretation of patient-reported outcomes (PROs) due to a multitude of instruments and measuring approaches. The aim of the study was to establish an expert consensus on the relevance and key indicators of quality of life in the clinical practice of breast cancer survivors. (2) Methods: Potential indicators of the quality of life of breast cancer survivors were extracted from the established quality of life models, depicting survivors' perspectives. The specific domains and subdomains of quality of life were evaluated in a two-stage online Delphi process, including an international and multidisciplinary panel of experts. (3) Results: The first round of the Delphi process was completed by 57 and the second by 37 participants. A consensus was reached for the Physical and Psychological domains, and on eleven subdomains of quality of life. The results were further supported by the additional ranking of importance of the subdomains in the second round. (4) Conclusions: The current findings can serve to optimize the use of instruments and address the challenges related to data collection and interpretation as the facilitators of the adaption in routine practice.

Discriminating Epithelial to Mesenchymal Transition Phenotypes in Circulating Tumor Cells Isolated from Advanced Gastrointestinal Cancer Patients.

Gastrointestinal (GI) cancers constitute a group of highest morbidity worldwide, with colorectal cancer (CRC) and gastric cancer being among the most frequently diagnosed. The majority of gastrointestinal cancer patients already present metastasis by the time of diagnosis, which is widely associated with cancer-related death. Accumulating evidence suggests that epithelial-to-mesenchymal transition (EMT) in cancer promotes circulating tumor cell (CTCs) formation, which ultimately drives metastasis development. These cells have emerged as a fundamental tool for cancer diagnosis and monitoring, as they reflect tumor heterogeneity and the clonal evolution of cancer in real-time. In particular, EMT phenotypes are commonly associated with therapy resistance. Thus, capturing these CTCs is expected to reveal important clinical information. However, currently available CTC isolation approaches are suboptimal and are often targeted to capture epithelial CTCs, leading to the loss of EMT or mesenchymal CTCs. Here, we describe size-based CTCs isolation using the RUBYchip™, a label-free microfluidic device, aiming to detect EMT biomarkers in CTCs from whole blood samples of GI cancer patients. We found that, for most cases, the mesenchymal phenotype was predominant, and in fact a considerable fraction of isolated CTCs did not express epithelial markers. The RUBYchip™ can overcome the limitations of label-dependent technologies and improve the identification of CTC subpopulations that may be related to different clinical outcomes.

Surface enhanced Raman spectroscopy for tumor nucleic acid: Towards cancer diagnosis and precision medicine.

Cancer is the second leading cause of death worldwide. Early diagnosis and personalization of treatment have become effective routes to control the increasing mortality rate. Since cancer is a genetic disease, there is a great demand for novel techniques to detect tumor nucleic acids (NAs) with increased sensitivity. In recent years, surface enhanced Raman spectroscopy (SERS) emerged as a popular technique for biosensing in cancer theranostics. Combined with molecular probes, SERS allows ultrasensitive and multiplex detection of tumor-derived NAs, with great potential for clinical cancer detection and subtyping. In this review, we summarize and compare the various strategies for designing SERS-based NA sensors, focusing on the mechanism of sensing, followed by their representative applications to cancer theranostics in recent 5 years, as well as future challenges for clinical translation. The review is aimed to provide basic guidelines for engineering SERS-based NA sensors, according to the specific clinical cancer application.

Use of some cost-effective technologies for a routine clinical pathology laboratory.

Classically, the need for highly sophisticated instruments with important economic costs has been a major limiting factor for clinical pathology laboratories, especially in developing countries. With the aim of making clinical pathology more accessible, a wide variety of free or economical technologies have been developed worldwide in the last few years. 3D printing and Arduino approaches can provide up to 94% economical savings in hardware and instrumentation in comparison to commercial alternatives. The vast selection of point-of-care-tests (POCT) currently available also limits the need for specific instruments or personnel, as they can be used almost anywhere and by anyone. Lastly, there are dozens of free and libre digital tools available in health informatics. This review provides an overview of the state-of-the-art on cost-effective alternatives with applications in routine clinical pathology laboratories. In this context, a variety of technologies including 3D printing and Arduino, lateral flow assays, plasmonic biosensors, and microfluidics, as well as laboratory information systems, are discussed. This review aims to serve as an introduction to different technologies that can make clinical pathology more accessible and, therefore, contribute to achieve universal health coverage.

HER2 Expression in Circulating Tumour Cells Isolated from Metastatic Breast Cancer Patients Using a Size-Based Microfluidic Device.

HER2 is a prognostic and predictive biomarker in breast cancer, normally assessed in tumour biopsy and used to guide treatment choices. Circulating tumour cells (CTCs) escape the primary tumour and enter the bloodstream, exhibiting great metastatic potential and representing a real-time snapshot of the tumour burden. Liquid biopsy offers the unique opportunity for low invasive sampling in cancer patients and holds the potential to provide valuable information for the clinical management of cancer patients. This study assesses the performance of the RUBYchip™, a microfluidic system for CTC capture based on cell size and deformability, and compares it with the only FDA-approved technology for CTC enumeration, CellSearch®. After optimising device performance, 30 whole blood samples from metastatic breast cancer patients were processed with both technologies. The expression of HER2 was assessed in isolated CTCs and compared to tissue biopsy. Results show that the RUBYchipTM was able to isolate CTCs with higher efficiency than CellSearch®, up to 10 times more, averaging all samples. An accurate evaluation of different CTC subpopulations, including HER2+ CTCs, was provided. Liquid biopsy through the use of the RUBYchipTM in the clinic can overcome the limitations of histological testing and evaluate HER2 status in patients in real-time, helping to tailor treatment during disease evolution.

Electrochemical Sensing in 3D Cell Culture Models: New Tools for Developing Better Cancer Diagnostics and Treatments.

Currently, conventional pre-clinical in vitro studies are primarily based on two-dimensional (2D) cell culture models, which are usually limited in mimicking the real three-dimensional (3D) physiological conditions, cell heterogeneity, cell to cell interaction, and extracellular matrix (ECM) present in living tissues. Traditionally, animal models are used to mimic the 3D environment of tissues and organs, but they suffer from high costs, are time consuming, bring up ethical concerns, and still present many differences when compared to the human body. The applications of microfluidic-based 3D cell culture models are advantageous and useful as they include 3D multicellular model systems (MCMS). These models have demonstrated potential to simulate the in vivo 3D microenvironment with relatively low cost and high throughput. The incorporation of monitoring capabilities in the MCMS has also been explored to evaluate in real time biophysical and chemical parameters of the system, for example temperature, oxygen, pH, and metabolites. Electrochemical sensing is considered as one of the most sensitive and commercially adapted technologies for bio-sensing applications. Amalgamation of electrochemical biosensing with cell culture in microfluidic devices with improved sensitivity and performance are the future of 3D systems. Particularly in cancer, such models with integrated sensing capabilities can be crucial to assess the multiple parameters involved in tumour formation, proliferation, and invasion. In this review, we are focusing on existing 3D cell culture systems with integrated electrochemical sensing for potential applications in cancer models to advance diagnosis and treatment. We discuss their design, sensing principle, and application in the biomedical area to understand the potential relevance of miniaturized electrochemical hybrid systems for the next generation of diagnostic platforms for precision medicine.

Single-use microfluidic device for purification and concentration of environmental DNA from river water.

Purification and concentration of DNA is a critical step on DNA-based analysis, which should ensure efficient DNA isolation and effective removal of contaminants that may interfere with downstream DNA amplification. Complexity of samples, minute content of target analyte, or high DNA fragmentation greatly entangles the success of this step. To overcome this issue, we designed and fabricated a novel miniaturized disposable device for a highly efficient DNA purification. The microfluidic device showed binding efficiency and elution yield of 90.1% and 86.7%, respectively. Moreover, the effect of DNA fragmentation, a parameter that has not been previously addressed, showed a great impact in the recovery step. The microfluidic system integrated micropillars with chitosan being used as the solid-phase for a pH-dependent DNA capture and release. We have showed the potential of the device in the successful purification of environmental DNA (eDNA) from river water samples contaminated with Dreissena polymorpha, an invasive alien species responsible for unquestionable economic and environmental consequences in river water basins. Additionally, the device was also able to concentrate the DNA extract from highly diluted samples, showing promising results for the early detection of such invasive species, which may allow prompt measures for a more efficient control in affected areas. Suitability for integration with downstream DNA analysis was also demonstrated through qPCR analysis of the samples purified with the microfluidic device, allowing detection of the target species even if highly diluted.

Target Score-A Proteomics Data Selection Tool Applied to Esophageal Cancer Identifies GLUT1-Sialyl Tn Glycoforms as Biomarkers of Cancer Aggressiveness.

Esophageal cancer (EC) is a life-threatening disease, demanding the discovery of new biomarkers and molecular targets for precision oncology. Aberrantly glycosylated proteins hold tremendous potential towards this objective. In the current study, a series of esophageal squamous cell carcinomas (ESCC) and EC-derived circulating tumor cells (CTCs) were screened by immunoassays for the sialyl-Tn (STn) antigen, a glycan rarely expressed in healthy tissues and widely observed in aggressive gastrointestinal cancers. An ESCC cell model was glycoengineered to express STn and characterized in relation to cell proliferation and invasion in vitro. STn was found to be widely present in ESCC (70% of tumors) and in CTCs in 20% of patients, being associated with general recurrence and reduced survival. Furthermore, STn expression in ESCC cells increased invasion in vitro, while reducing cancer cells proliferation. In parallel, an ESCC mass spectrometry-based proteomics dataset, obtained from the PRIDE database, was comprehensively interrogated for abnormally glycosylated proteins. Data integration with the Target Score, an algorithm developed in-house, pinpointed the glucose transporter type 1 (GLUT1) as a biomarker of poor prognosis. GLUT1-STn glycoproteoforms were latter identified in tumor tissues in patients facing worst prognosis. Furthermore, healthy human tissues analysis suggested that STn glycosylation provided cancer specificity to GLUT1. In conclusion, STn is a biomarker of worst prognosis in EC and GLUT1-STn glycoforms may be used to increase its specificity on the stratification and targeting of aggressive ESCC forms.

Phenotypic Analysis of Urothelial Exfoliated Cells in Bladder Cancer via Microfluidic Immunoassays: Sialyl-Tn as a Novel Biomarker in Liquid Biopsies.

Bladder cancer is the most common malignancy of the urinary tract, having one of the highest recurrence rates and progression from non-muscle to muscle invasive bladder cancer that commonly leads to metastasis. Cystoscopy and urine cytology are the standard procedures for its detection but have limited clinical sensitivity and specificity. Herein, a microfluidic device, the UriChip, was developed for the enrichment of urothelial exfoliated cells from fresh and frozen urine, based on deformability and size, and the cancer-associated glycan Sialyl-Tn explored as a putative bladder cancer urinary biomarker. Spiking experiments with bladder cancer cell lines showed an isolation efficiency of 53%, while clinical sample analyses revealed retention of cells with various morphologies and sizes. in situ immunoassays demonstrated significantly higher number of Sialyl-Tn-positive cells in fresh and frozen voided urine from bladder cancer patients, compared to healthy individuals. Of note, urothelial exfoliated cells from cryopreserved urine sediments were also successfully isolated by the UriChip, and found to express significantly high levels of Sialyl-Tn. Remarkably, Sialyl-Tn expression is correlated with tumor stage and grade. Overall, our findings demonstrate the potential of UriChip and Sialyl-Tn to detect urothelial bladder cancer cells in follow-up and long-term retrospective studies.

Profiling DNA mutation patterns by SERS fingerprinting for supervised cancer classification.

Profiling DNA mutation patterns for cancer classification plays an essential role in precision and personalized medicine. Conventional PCR-based mutation assay is limited by the extensive labour on target amplification. We herein create an amplification-free surface enhanced Raman spectroscopy (SERS) biochip which enables direct and simultaneous identification of multiple point mutations in tumor cells. Without pre-amplifying the target sequences, the SERS assay reads out the presence of cellular mutations through the interpretation of Raman fingerprints. The SERS sensor is integrated into a microfluidic chip, achieving one-step multiplex analysis within 40 min. Importantly, by combining SERS spectra encoding technique with supervised learning algorithm, a panel of nucleotide mixtures can be well distinguished according to their mutation profiles. We initially demonstrate an excellent levels of classification in samples from colorectal cancer and melanoma cell lines. For final clinical validation, the system performance is verified by classifying cancer patient samples, which shows an accuracy above 90%. Due to the simplicity and rapidness, the SERS biosensor is expected to become a promising tool for clinical point-of-care diagnosis towards precision medicine.

In Vitro Evaluation of Lipopolyplexes for Gene Transfection: Comparing 2D, 3D and Microdroplet-Enabled Cell Culture.

Complexes combining nucleic acids with lipids and polymers (lipopolyplexes) show great promise for gene therapy since they enable compositional, physical and functional versatility to be optimized for therapeutic efficiency. When developing lipopolyplexes for gene delivery, one of the first evaluations performed is an in vitro transfection efficiency experiment. Many different in vitro models can be used, and the effect of the model on the experiment outcome has not been thoroughly studied. The objective of this work was to compare the insights obtained from three different in vitro models, as well as the potential limitations associated with each of them. We have prepared a series of lipopolyplex formulations with three different cationic polymers (poly-l-lysine, bioreducible poly-l-lysine and polyethyleneimine), and assessed their in vitro biological performance in 2D monolayer cell culture, 3D spheroid culture and microdroplet-based single-cell culture. Lipopolyplexes from different polymers presented varying degrees of transfection efficiency in all models. The best-performing formulation in 2D culture was the polyethyleneimine lipopolyplex, while lipoplexes prepared with bioreducible poly-l-lysine were the only ones achieving any transfection in microdroplet-enabled cell culture. None of the prepared formulations achieved significant gene transfection in 3D culture. All of the prepared formulations were well tolerated by cells in 2D culture, while at least one formulation (poly-l-lysine polyplex) delayed 3D spheroid growth. These results highlight the need for selecting the appropriate in vitro model depending on the intended application.

Multifuntional Gold Nanoparticles for the SERS Detection of Pathogens Combined with a LAMP-in-Microdroplets Approach.

We developed a droplet-based optofluidic system for the detection of foodborne pathogens. Specifically, the loop-mediated isothermal amplification (LAMP) technique was combined with surface-enhanced Raman scattering (SERS), which offers an excellent method for DNA ultradetection. However, the direct SERS detection of DNA compromises the simplicity of data interpretation due to the variability of its SERS fingerprints. Therefore, we designed an indirect SERS detection method using multifunctional gold nanoparticles (AuNPs) based on the formation of pyrophosphate generated during the DNA amplification by LAMP. Towards this goal, we prepared multifunctional AuNPs involving three components with key roles: (1) thiolated poly(ethylene glycol) as stabilizing agent, (2) 1-naphthalenethiol as Raman reporter, and (3) glutathione as a bioinspired chelating agent of magnesium (II) ions. Thus, the variation in the SERS signal of 1-naphthalenethiol was controlled by the aggregation of AuNPs triggered by the complexation of pyrophosphate and glutathione with free magnesium ions. Using this strategy, we detected Listeria monocytogenes, not only in buffer, but also in a food matrix (i.e., ultra-high temperaturemilk) enabled by the massive production of hotspots as a result of the self-assemblies that enhanced the SERS signal. This allowed the development of a microdroplet-LAMP-SERS platform with isothermal amplification and real-time identification capabilities.

Portable sensing system based on electrochemical impedance spectroscopy for the simultaneous quantification of free and total microcystin-LR in freshwaters.

Microcystins are the most worldwide extended and common toxins produced by cyanobacteria in freshwater. Microcystin-leucine arginine (MC-LR), associated with the most toxic incidents involving microcystins, are within the cyanobacteria (intracellular) until released into the surrounding waters (extracellular) during cell lysis. Therefore, the relationship between intracellular and extracellular cyanotoxins will allow a comprehensive risk of cyanobacteria-containing waters, preventing disease and improving human safety. In this work, we present the development of a novel portable microfluidic sensing platform for the simultaneous detection of free (extracellular) and total MC-LR (intracellular and extracellular). The integrated system contains the sample processing and detection modules capable of performing the chemical lysis, filtration, sample mixing with antibodies, and electrochemical detection of MC-LR based on an indirect strategy. The performance of the immunosensors was evaluated by electrochemical impedance spectroscopy, showing a linear dynamic range between 3.3 × 10-4 and 10-7 g L-1 and a limit of detection of 5.7 × 10-10 g L-1. The results demonstrate the potential of the developed portable biosensor platform and its suitable application for the analysis of MC-LR at regulated levels for drinking water. Finally, the integrated system was able to simultaneously detect the free and total MC-LR on a Microcystis aeruginosa culture. To the best of our knowledge this is the first described system that can differentiate between intracellular and extracellular concentration of MC-LR. This novel electrochemical sensing platform avoids the multiple processing steps typically needed for standard MC-LR analysis in the laboratory and provides an early warning system for MC-LR remote monitoring in water.