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We introduce a novel approach for highly parallel droplet dispensing with precise control over the droplet parameters such as droplet volume, droplet velocity, etc. This approach facilitates the fabrication of homogeneous and precise thin layers with uniform coverage on defined small areas (e.g., a specific area of 1 × 1.4 mm2 in microfluidic channels or microwells). The presented approach ensures layer uniformity and high precision in X/Y extent and edge resolution, making it well suited for achieving precise and controlled coating for a variety of applications such as homogeneous coatings for lateral flow tests, ELISA plates, and biosensors for continuous glucose monitoring (CGM) devices. Our approach is based on direct liquid displacement employing a piston that is in direct contact with the liquid and an array of nozzles. Considering a variety of nozzle chip designs (i.e., varying nozzle diameter and pitch), we evaluated a multitude of parameters to derive general design rules for the nozzle chip design. Thus, we achieved a tunable droplet volume from 200 to 800 pL and droplet velocities from 0.5 to 2.5 m/s, applying a nozzle diameter of 50 µm and a nozzle pitch of 165 µm. The presented results showcase the versatility of the approach, offering precise dispensing capabilities.
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The technology to jet print metal lines with precise shape fidelity on diverse substrates is gaining higher interest across multiple research fields. It finds applications in additively manufactured flexible electronics, environmentally friendly and sustainable electronics, sensor devices for medical applications, and fabricating electrodes for solar cells. This paper provides an experimental investigation to deepen insights into the non-contact printing of solder lines using StarJet technology, eliminating the need for surface activation, substrate heating, curing, or post-processing. Moreover, it employs bulk metal instead of conventional inks or pastes, leading to cost-effective production and enhanced conductivity. The effect of molten metal temperature, substrate temperature, standoff distance, and printing velocity was investigated on polymer foils (i.e., PET sheets). Robust printing parameters were derived to print uniform, bulge-free, bulk metal lines suitable for additive manufacturing applications. The applicability of the derived parameters was extended to 3D-printed PLA, TPU, PA-GF, and PETG substrates having a much higher surface roughness. Additionally, a high aspect ratio of approx. 16:1 wall structure has been demonstrated by printing multiple metal lines on top of each other. While challenges persist, this study contributes to advancing additively manufactured electronic devices, highlighting the capabilities of StarJet metal jet-printing technology.
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In this article, we explore multi-material additive manufacturing (MMAM) for conductive trace printing using molten metal microdroplets on polymer substrates to enhance digital signal transmission. Investigating microdroplet spread informs design rules for adjacent trace printing. We studied the effects of print distance on trace morphology and resolution, noting that printing distance showed almost no change in the printed trace pitch. Crosstalk interference between adjacent signal traces was analyzed across frequencies and validated both experimentally and through simulation; no crosstalk was visible for printed traces at input frequencies below 600 kHz. Moreover, we demonstrate printed trace reliability against thermal shock, whereby no discontinuation in conductive traces was observed. Our findings establish design guidelines for MMAM electronics, advancing digital signal transmission capabilities.
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Creating model systems that replicate in vivo tissues is crucial for understanding complex biological pathways like drug response and disease progression. Three-dimensional (3D) in vitro models, especially multicellular spheroids (MCSs), offer valuable insights into physiological processes. However, generating MCSs at scale with consistent properties and efficiently recovering them pose challenges. We introduce a workflow that automates large-scale spheroid production and enables parallel harvesting into individual wells of a microtiter plate. Our method, based on the hanging-drop technique, utilizes a non-contact dispenser for dispensing nanoliter droplets of a uniformly mixed-cell suspension. The setup allows for extended processing times of up to 45 min without compromising spheroid quality. As a proof of concept, we achieved a 99.3% spheroid generation efficiency and maintained highly consistent spheroid sizes, with a coefficient of variance below 8% for MCF7 spheroids. Our centrifugation-based drop transfer for spheroid harvesting achieved a sample recovery of 100%. We successfully transferred HT29 spheroids from hanging drops to individual wells preloaded with collagen matrices, where they continued to proliferate. This high-throughput workflow opens new possibilities for prolonged spheroid cultivation, advanced downstream assays, and increased hands-off time in complex 3D cell culture protocols.
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3D cell culture is becoming increasingly important for mimicking physiological tissue structures in areas such as drug discovery and personalized medicine. To enable reproducibility on a large scale, automation technologies for standardized handling are still a challenge. Here, a novel method for fully automated size classification and handling of cell aggregates like spheroids and organoids is presented. Using microfluidic flow generated by a piezoelectric droplet generator, aggregates are aspirated from a reservoir on one side of a thin capillary and deposited on the other side, encapsulated in free-flying nanoliter droplets to a target. The platform has aggregate aspiration and plating efficiencies of 98.1% and 98.4%, respectively, at a processing throughput of up to 21 aggregates per minute. Cytocompatibility of the method is thoroughly assessed with MCF7, LNCaP, A549 spheroids and colon organoids, revealing no adverse effects on cell aggregates as shear stress is reduced compared to manual pipetting. Further, generic size-selective handling of heterogeneous organoid samples, single-aggregate-dispensing efficiencies of up to 100% and the successful embedding of spheroids or organoids in a hydrogel with subsequent proliferation is demonstrated. This platform is a powerful tool for standardized 3D in vitro research.
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Microfluídica , Organoides , Reproducibilidad de los Resultados , Automatización , Microfluídica/métodos , Técnicas de Cultivo Tridimensional de Células , Esferoides CelularesRESUMEN
C-reactive protein is a well-studied host response biomarker, whose diagnostic performance depends on its accurate classification into concentration zones defined by clinical scenario-specific cutoff values. We validated a newly developed, bead-based, bound-free phase detection immunoassay (BFPD-IA) versus a commercial CE-IVD enzyme-linked immunosorbent assay (ELISA) kit and a commercial CE-IVD immunoturbidimetric assay (ITA) kit. The latter was performed on a fully automated DPC Konelab 60i clinical analyzer used in routine diagnosis. We classified 53 samples into concentration zones derived from four different sets of cutoff values that are related to antibiotic prescription scenarios in the case of respiratory tract infections. The agreements between the methods were ELISA/ITA at 87.7%, ELISA/BFPD-IA at 87.3%, and ITA/-BFPD-IA at 93.9%, reaching 98-99% in all cases when considering the calculated relative combined uncertainty of the single measurement of each sample. In a subgroup of 37 samples, which were analyzed for absolute concentration quantification, the scatter plot slopes' correlations were as follows: ELISA/ITA 1.15, R2 = 0.97; BFPD-IA/ELISA 1.12, R2 = 0.95; BFPD-IA/ITA 0.95, R2 = 0.93. These very good performances and the agreement between BFPD-IA and ITA (routine diagnostic), combined with BFPD-IA's functional advantages over ITA (and ELISA)-such as quick time to result (~20 min), reduced consumed reagents (only one assay buffer and no washing), few and easy steps, and compatibility with nucleic-acid-amplification instruments-render it a potential approach for a reliable, cost-efficient, evidence-based point-of-care diagnostic test for guiding antibiotic prescriptions.
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Proteína C-Reactiva , Humanos , Proteína C-Reactiva/análisis , Inmunoensayo/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , BiomarcadoresRESUMEN
Globally, tuberculosis (TB) remains the deadliest bacterial infectious disease, and spreading antibiotic resistances is the biggest challenge for combatting the disease. Rapid and comprehensive diagnostics including drug susceptibility testing (DST) would assure early treatment, reduction of morbidity and the interruption of transmission chains. To date, rapid genetic resistance testing addresses only one to four drug groups while complete DST is done phenotypically and takes several weeks. To overcome these limitations, we developed a two-stage workflow for rapid TB diagnostics including DST from a single sputum sample that can be completed within three days. The first stage is qPCR detection of M. tuberculosis complex (MTBC) including antibiotic resistance testing against the first-line antibiotics, isoniazid (Inh) and rifampicin (Rif). The test is automated by centrifugal microfluidics and designed for point of care (PoC). Furthermore, enriched MTBC DNA is provided in a detachable sample tube to enable the second stage: if the PCR detects MTBC and resistance to either Inh or Rif, the MTBC DNA is shipped to specialized facilities and analyzed by targeted next generation sequencing (tNGS) to assess the complete resistance profile. Proof-of-concept testing of the PoC test revealed an analytical sensitivity of 44.2 CFU ml-1, a diagnostic sensitivity of 96%, and a diagnostic specificity of 100% for MTBC detection. Coupled tNGS successfully provided resistance profiles, demonstrated for samples from 17 patients. To the best of our knowledge, the presented combination of PoC qPCR with tNGS allows for the fastest comprehensive TB diagnostics comprising decentralized pathogen detection with subsequent resistance profiling in a facility specialized in tNGS.
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Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Tuberculosis , Humanos , Rifampin/farmacología , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Mycobacterium tuberculosis/genética , Pruebas de Sensibilidad Microbiana , Sistemas de Atención de Punto , Microfluídica , Sensibilidad y Especificidad , Tuberculosis/diagnóstico , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , Isoniazida/farmacología , Isoniazida/uso terapéutico , Farmacorresistencia Microbiana , ADNRESUMEN
Highly effective targeted therapies are available to treat noncommunicable chronic inflammatory skin diseases. In contrast, the exact diagnosis of noncommunicable chronic inflammatory skin diseases is complicated by its complex pathogenesis and clinical and histological overlap. Particularly, the differential diagnosis of psoriasis and eczema can be challenging in some cases, and molecular diagnostic tools need to be developed to support a gold standard diagnosis. The aim of this work was to develop a real-time PCR-based molecular classifier to distinguish psoriasis from eczema in formalin-fixed and paraffin-embedded-fixed skin samples and to evaluate the use of minimally invasive microbiopsies and tape strips for molecular diagnosis. In this study, we present a formalin-fixed and paraffin-embedded-based molecular classifier that determines the probability for psoriasis with a sensitivity/specificity of 92%/100%, respectively, and an area under the curve of 0.97, delivering comparable results to our previous published RNAprotect-based molecular classifier. The psoriasis probability, as well as levels of NOS2 expression, positively correlated with the disease hallmarks of psoriasis and negatively with eczema hallmarks. Furthermore, minimally invasive tape strips and microbiopsies were effectively used to differentiate psoriasis from eczema. In summary, the molecular classifier offers broad usage in pathology laboratories as well as outpatient settings and can support the differential diagnosis of noncommunicable chronic inflammatory skin diseases on a molecular level using formalin-fixed and paraffin-embedded tissue, microbiopsies, and tape strips.
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Eccema , Psoriasis , Humanos , Formaldehído , Fijación del Tejido/métodos , Diagnóstico Diferencial , Adhesión en Parafina , Psoriasis/diagnóstico , Psoriasis/genética , Psoriasis/metabolismo , Eccema/diagnóstico , Eccema/genética , Expresión GénicaRESUMEN
cfDNA is an emerging biomarker with promising uses for the monitoring of cancer or infectious disease diagnostics. This work demonstrates a new concept for an automated cfDNA extraction with nanobeads as the solid phase in a centrifugal microfluidic LabDisk. By using a combination of centrifugal and magnetic forces, we retain the nanobeads in one incubation chamber while sequentially adding, incubating and removing the sample and pre-stored buffers for extraction. As the recovery rate of the typically low concentration of cfDNA is of high importance to attain sufficient amounts for analysis, optimal beadhandling is paramount. The goal is that the cfDNA in the sample adsorbs to the solid phase completely during the binding step, is retained during washing and completely removed during elution. In this work, we improved beadhandling by optimizing the incubation chamber geometry and both frequency and temperature protocols, to maximize recovery rates. For characterization of the extraction performance, synthetic mutant DNA was spiked into human plasma samples. The LabDisk showed better reproducibility in DNA recovery rates with a standard deviation of ±13% compared to a manual approach using spin-columns (±17%) or nanobeads (±26%). The extraction of colorectal cancer samples with both the developed LabDisk and a robotic automation instrument resulted in comparable allele frequencies. Consequently, we present a highly attractive solution for an automated liquid biopsy cfDNA extraction in a small benchtop device.
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Ácidos Nucleicos Libres de Células , Biopsia Líquida , Microfluídica , Humanos , Ácidos Nucleicos Libres de Células/genética , ADN/genética , Microfluídica/métodos , Reproducibilidad de los Resultados , Nanoestructuras , CentrifugaciónRESUMEN
Spheroids, organoids, or cell-laden droplets are often used as building blocks for bioprinting, but so far little is known about the spatio-temporal cellular interactions subsequent to printing. We used a drop-on-demand bioprinting approach to study the biological interactions of such building blocks in dimensions of micrometers. Highly-density droplets (approximately 700 cells in 10 nL) of multiple cell types were patterned in a 3D hydrogel matrix with a precision of up to 70 µm. The patterns were used to investigate interactions of endothelial cells (HUVECs) and adipose-derived mesenchymal stem cells (ASCs), which are related to vascularization. We demonstrated that a gap of 200 µm between HUVEC and ASC aggregates led to decreased sprouting of HUVECs towards ASCs and increased growth from ASCs towards HUVECs. For mixed aggregates containing both cell types, cellular interconnections of ASCs with lengths of up to approximately 800 µm and inhibition of HUVEC sprouting were observed. When ASCs were differentiated into smooth muscle cells (dASCs), separate HUVEC aggregates displayed decreased sprouting towards dASCs, whereas no cellular interconnections nor inhibition of HUVEC sprouting were detected for mixed dASCs/HUVEC aggregates. These findings demonstrate that our approach could be applied to investigate cell-cell interactions of different cell types in 3D co-cultures.
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Bioimpresión , Células Madre Mesenquimatosas , Humanos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Bioimpresión/métodos , Células Madre Mesenquimatosas/metabolismo , Comunicación Celular , Hidrogeles/farmacologíaRESUMEN
Accurately measuring flow rates is a key requirement in many medical applications such as infusion and drug delivery systems. A major drawback of current systems is the low resolution of the sensors in the low flow rate regime. In this article, we present a method based on Holographic PIV/PTV that has been used for the first time to measure flow rates in the range of a few nL/min. Our method requires a very simple setup that combines lensless holography with particle velocimetry. For flow rates in the 70 to 200 nL/min range, the highest uncertainty was 5.6% (coverage factor k=2). This is an open-source project; the CAD designs and software source code are available at https://github.com/gui-miotto/holovel.
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Holografía , Reología/métodos , Sistemas de Liberación de MedicamentosRESUMEN
Centrifugal microfluidics enables fully automated molecular diagnostics at the point-of-need. However, the integration of solid-phase nucleic acid extraction remains a challenge. Under this scope, we developed the magnetophoresis under continuous rotation for magnetic bead-based nucleic acid extraction. Four stationary permanent magnets are arranged above a cartridge, creating a magnetic field that enables the beads to be transported between the chambers of the extraction module under continuous rotation. The centrifugal force is maintained to avoid uncontrolled spreading of liquids. We concluded that below a frequency of 5 Hz, magnetic beads move radially inwards. In support of magnetophoresis, bead inertia and passive geometrical design features allow to control the azimuthal bead movement between chambers. We then demonstrated ferrimagnetic bead transfer in liquids with broad range of surface tension and density values. Furthermore, we extracted nucleic acids from lysed Anopheles gambiae mosquitoes reaching comparable results of eluate purity (LabDisk: A260/A280 = 1.6 ± 0.04; Reference: 1.8 ± 0.17), and RT-PCR of extracted RNA (LabDisk: Ct = 17.9 ± 1.6; Reference: Ct = 19.3 ± 1.7). Conclusively, magnetophoresis at continuous rotation enables easy cartridge integration and nucleic acid extraction at the point-of-need with high yield and purity.
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Three-dimensional (3D) cell agglomerates, such as microtissues, organoids, and spheroids, become increasingly relevant in biomedicine. They can provide in vitro models that recapitulate functions of the original tissue in the body and have applications in cancer research. For example, they are widely used in organ-on-chip systems. Microsensors can provide essential real-time information on cell metabolism as well as the reliability and quality of culture conditions. The combination of sensors and 3D cell cultures, especially single spheroids, is challenging in terms of reproducible formation, manipulation, and access to spheroids, precise positioning near sensors, and high cell-to-volume ratios to obtain meaningful biosignals in the most parallel approach possible. To overcome this challenge, we combined state-of-the-art bioprinting techniques to automatically print tumour spheroids directly into microwells of a chip-based electrochemical oxygen sensor array. We demonstrated highly accurate and reproducible spheroid formation (diameter of approx. 200 µm) and a spheroid deposition precision of 25 µm within a volume of 22 nl per droplet. Microstructures and hydrogel-coated microwells allowed the placement of single MCF-7 breast cancer spheroids close to the sensor electrodes. The microelectrode wells were sealed for oxygen measurements within a 55 nl volume for fast concentration changes. Accurate and stable amperometric oxygen sensor performance was demonstrated from atmospheric to anoxic regions. Cellular respiration rates from single tumour spheroids in the range of 450-850 fmol min-1 were determined, and alterations of cell metabolism upon drug exposure were shown. Our results uniquely combine bioprinting with 3D cell culture monitoring and demonstrate the much-needed effort for facilitation, parallelization, sensor integration, and drug delivery in 3D cell culture and organ-on-chip experiments. The workflow has a high degree of automation and potential for scalability. In order to achieve greater flexibility in the automation of spheroid formation and trapping, we employed a method based on drop-on-demand liquid handling systems, instead of the typical on-chip approach commonly used in microfluidics. Its relevance ranges from fundamental metabolic research over standardization of cell culture experiments and toxicological studies, to personalized medicine, e.g. patient-specific chemotherapy.
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Bioimpresión , Neoplasias , Humanos , Bioimpresión/métodos , Esferoides Celulares , Microelectrodos , Reproducibilidad de los Resultados , OxígenoRESUMEN
In this paper, we present the ImmunoDisk, a fully automated sample-to-answer centrifugal microfluidic cartridge, integrating a heterogeneous, wash-free, magnetic- and fluorescent bead-based immunoassay (bound-free phase detection immunoassay/BFPD-IA). The BFPD-IA allows the implementation of a simple fluidic structure, where the assay incubation, bead separation and detection are performed in the same chamber. The system was characterized using a C-reactive protein (CRP) competitive immunoassay. A parametric investigation on air drying of protein-coupled beads for pre-storage at room temperature is presented. The key parameters were buffer composition, drying temperature and duration. A protocol for drying two different types of protein-coupled beads with the same temperature and duration using different drying buffers is presented. The sample-to-answer workflow was demonstrated measuring CRP in 5 µL of human serum, without prior dilution, utilizing only one incubation step, in 20 min turnaround time, in the clinically relevant concentration range of 15-115 mg/L. A reproducibility assessment over three disk batches revealed an average signal coefficient of variation (CV) of 5.8 ± 1.3%. A CRP certified reference material was used for method verification with a concentration CV of 8.6%. Our results encourage future testing of the CRP-ImmunoDisk in clinical studies and its point-of-care implementation in many diagnostic applications.
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Proteína C-Reactiva , Microfluídica , Humanos , Inmunoensayo/métodos , Indicadores y Reactivos , Reproducibilidad de los ResultadosRESUMEN
We used arrays of bioprinted renal epithelial cell spheroids for toxicity testing with cisplatin. The concentration-dependent cell death rate was determined using a lactate dehydrogenase assay. Bioprinted spheroids showed enhanced sensitivity to the treatment in comparison to monolayers of the same cell type. The measured dose-response curves revealed an inhibitory concentration of the spheroids of IC 50 = 9 ± 3 µM in contrast to the monolayers with IC 50 = 17 ± 2 µM. Fluorescent labeling of a nephrotoxicity biomarker, kidney injury molecule 1 indicated an accumulation of the molecule in the central lumen of the spheroids. Finally, we tested an approach for an automatic readout of toxicity based on microscopic images with deep learning. Therefore, we created a dataset comprising images of single spheroids, with corresponding labels of the determined cell death rates for training. The algorithm was able to distinguish between three classes of no, mild, and severe treatment effects with a balanced accuracy of 78.7%.
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We demonstrate detection and quantification of bacterial load with a novel microfluidic one-pot wash-free fluorescence in situ hybridization (FISH) assay in droplets. The method offers minimal manual workload by only requiring mixing of the sample with reagents and loading it into a microfluidic cartridge. By centrifugal microfluidic step emulsification, our method partitioned the sample into 210 pL (73 µm in diameter) droplets for bacterial encapsulation followed by in situ permeabilization, hybridization, and signal detection. Employing locked nucleic acid (LNA)/DNA molecular beacons (LNA/DNA MBs) and NaCl-urea based hybridization buffer, the assay was characterized with Escherichia coli, Klebsiella pneumonia, and Proteus mirabilis. The assay performed with single-cell sensitivity, a 4-log dynamic range from a lower limit of quantification (LLOQ) at ~3 × 103 bacteria/mL to an upper limit of quantification (ULOQ) at ~3 × 107 bacteria/mL, anda linearity R2 = 0.976. The total time-to-results for detection and quantification was around 1.5 hours.
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ADN , Microfluídica , Escherichia coli/genética , Hibridación Fluorescente in Situ/métodos , Microfluídica/métodos , OligonucleótidosRESUMEN
The generation of artificial human tissue by 3D-bioprinting has expanded significantly as a clinically relevant research topic in recent years. However, to produce a complex and viable tissue, in-depth biological understanding and advanced printing techniques are required with a high number of process parameters. Here, we systematically evaluate the process parameters relevant for a hybrid bioprinting process based on fused-deposition modeling (FDM) of thermoplastic material and microextrusion of a cell-laden hydrogel. First, we investigated the effect of the printing temperature of polycaprolactone (PCL), on the junction strength between individual fused filaments and on the viability of immortalized mesenchymal stem cells (iMSC) in the surrounding alginate-gelatin-hydrogel. It was found that a printing temperature of 140 °C and bonds with an angle of 90° between the filaments provided a good compromise between bonding strength of the filaments and the viability of the surrounding cells. Using these process parameters obtained from individual fused filaments, we then printed cubic test structures with a volume of 10 × 10 × 10 mm3 with different designs of infill patterns. The variations in mechanical strength of these cubes were measured for scaffolds made of PCL-only as well as for hydrogel-filled PCL scaffolds printed by alternating hybrid bioprinting of PCL and hydrogel, layer by layer. The bare scaffolds showed a compressive modulus of up to 6 MPa, close to human hard tissue, that decreased to about 4 MPa when PCL was printed together with hydrogel. The scaffold design suited best for hybrid printing was incubated with cell-laden hydrogel and showed no degradation of its mechanical strength for up to 28 days.
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Bioimpresión , Alginatos , Bioimpresión/métodos , Gelatina , Humanos , Hidrogeles , Poliésteres , Impresión Tridimensional , Ingeniería de Tejidos/métodos , Andamios del Tejido/químicaRESUMEN
Protein electrophoresis and immunoblotting are indispensable analytical tools for the characterization of proteins and posttranslational modifications in complex sample matrices. Owing to the lack of automation, commonly employed slab-gel systems suffer from high time demand, significant sample/antibody consumption, and limited reproducibility. To overcome these limitations, we developed a paper-based open microfluidic platform for electrophoretic protein separation and subsequent transfer to protein-binding membranes for immunoprobing. Electrophoresis microstructures were digitally printed into cellulose acetate membranes that provide mechanical stability while maintaining full accessibility of the microstructures for consecutive immunological analysis. As a proof-of-concept, we demonstrate separation of fluorescently labeled marker proteins in a wide molecular weight range (15-120 kDa) within only 15 min, reducing the time demand for the entire workflow (from sample preparation to immunoassay) to approximately one hour. Sample consumption was reduced 10- to 150-fold compared to slab-gel systems, owing to system miniaturization. Moreover, we successfully applied the paper-based approach to complex samples such as crude bacterial cell extracts. We envisage that this platform will find its use in protein analysis workflows for scarce and precious samples, providing a unique opportunity to extract profound immunological information from limited sample amounts in a fast fashion with minimal hands-on time.
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Técnicas Analíticas Microfluídicas , Microfluídica , Electroforesis , Immunoblotting , Microfluídica/métodos , Proteínas , Reproducibilidad de los ResultadosRESUMEN
Acute lymphoblastic leukemia (ALL) is the most frequent malignancy in childhood. Minimal residual disease (MRD) monitoring is an important prognostic factor for ALL treatment response and patient stratification. MRD monitoring uses personalized real-time PCR to measure the amount of cancer cells among normal cells. Due to clonal tumor evolution or secondary rearrangement processes, MRD markers can disappear during treatment, leading to false-negative MRD results and wrong decision-making in personalized treatments. Therefore, monitoring of multiple MRD markers per patient is required. For the first time, the authors present personalized multiplex mediator probe PCR (MP PCR) for MRD monitoring in ALL. These assays can precisely quantify more MRD markers in less sample material. Therefore, clinical outcomes will be less affected by clonal tumor evolution. Personalized duplex MP PCR assays were developed for different genomic MRD markers, including immunoglobulin/T-cell receptor gene rearrangements, gene fusions, and gene deletions. One duplex assay was successfully applied in a prospective patient case and compared with hydrolysis probes. Moreover, the authors increased the multiplex level from duplex to 4-plex and still met the EuroMRD requirements for reliable quantification. In addition, the authors' MRD-MP design guidelines and multiplex workflow facilitate and accelerate MP PCR assay development. This helps the standardization of personal diagnostics.
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Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Reacción en Cadena de la Polimerasa Multiplex , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Estudios Prospectivos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Periodontitis and dental caries are two major bacterially induced, non-communicable diseases that cause the deterioration of oral health, with implications in patients' general health. Early, precise diagnosis and personalized monitoring are essential for the efficient prevention and management of these diseases. Here, we present a disk-shaped microfluidic platform (OralDisk) compatible with chair-side use that enables analysis of non-invasively collected whole saliva samples and molecular-based detection of ten bacteria: seven periodontitis-associated (Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola) and three caries-associated (oral Lactobacilli, Streptococcus mutans, Streptococcus sobrinus). Each OralDisk test required 400 µL of homogenized whole saliva. The automated workflow included bacterial DNA extraction, purification and hydrolysis probe real-time PCR detection of the target pathogens. All reagents were pre-stored within the disk and sample-to-answer processing took < 3 h using a compact, customized processing device. A technical feasibility study (25 OralDisks) was conducted using samples from healthy, periodontitis and caries patients. The comparison of the OralDisk with a lab-based reference method revealed a ~90% agreement amongst targets detected as positive and negative. This shows the OralDisk's potential and suitability for inclusion in larger prospective implementation studies in dental care settings.