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Adaptations of new generation molecular techniques for multiplexed detection of pathogens are gaining interest in the field of point-of-care (POC) industry and onsite testing. Loop-mediated isothermal amplification (LAMP), an advanced molecular amplification technique, has proven promising due to its unique features that suits ideal for POC applications. However, application of LAMP for multiplexed detection of pathogens remains challenging because of the difficulty in the identification of specific LAMP amplicons that does not have a well-definite molecular size. In this study, we developed a solid-phase loop-mediated isothermal amplification (SP-LAMP) technique to address the challenge. Integration of LAMP with the supercritical angle fluorescence (SAF) micro-optic structures as a solid support (SS) in an array format enabled spatial separation of LAMP amplicons in a multiplexed configuration. Important parameters such as length of the SS primers, length of the primer-binding region, the effect of surface density of immobilized SS primers, and cross-reactivity among the primers of different targets were iteratively tested and optimized. With the combination of SP-LAMP and SAF techniques, it was possible to detect multiple pathogens that include

The COVID-19 pandemic emphasized the importance of rapid, portable, and on-site testing technologies necessary for resource-limited settings for effective testing and screening to reduce spreading of the infection. Realizing this, we developed a fluorescence-based point-of-care (fPOC) detection system with real-time reverse transcriptase loop-mediated isothermal amplification for rapid and quantitative detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The system is built based on the Arduino platform compatible with commercially available open-source hardware–software and off-the-shelf electronic components. The fPOC system comprises of three main components: 1) an instrument with integrated heaters, 2) optical detection components, and 3) an injection-molded polymeric cartridge. The system was tested and experimentally proved to be able to use for fast detection of the SARS-CoV-2 virus in real-time in less than 30 min. Preliminary results of testing the performance of the fPOC revealed that the fPOC could detect the SARS-CoV-2 virus at a limit of detection (LOD50%) at two to three copies/microliter (15.36 copies/reaction), which was comparable to reactions run on a standard commercial thermocycler. The performance of the fPOC was evaluated with 12 SARS-CoV-2 clinical throat swab samples that included seven positive and five negative samples, as confirmed by reverse transcription–polymerase chain reaction. The fPOC showed 100% agreement with the commercial thermocycler. This simple design of the fPOC system demonstrates the potential to greatly enhance the practical applicability to develop a totally integrated point-of-care system for rapid on-site screening of the SARS-CoV-2 virus in the management of the pandemic.

Invasive non-typhoidal salmonellosis is gaining worldwide attention as an emerging disease cluster among bloodstream infections. The disease has the highest burden among immunocompromised and malnourished children in resource-limited areas due to poor access to reliable and rapid diagnostics. Point-of-care (POC) diagnostics are promising for use in such low infrastructure laboratory settings. However, there still remains a major challenge for POC testing to deal with the complexity of blood matrices in rapid detection of an extremely low concentration of blood-borne pathogens. In this work, the challenges were addressed by combining magnetic bead based pathogen concentration and Loop Mediated Isothermal Amplification (LAMP) technology. Sensitivity and performance of the combined approach were determined and compared with a direct PCR method. A direct visual detection strategy, adapted using SYTO-24 DNA intercalating dye, resulted in a limit of detection (LoD) as low as 14 CFU/mL in blood samples with a total analysis time of less than 2 h, including sample preparation. This approach has the potential for wide application as a high-throughput POC testing method to analyze pathogens in clinical, food, feed and environmental samples.

Avian influenza virus (AIV) outbreaks occur frequently worldwide, causing a potential public health risk and great economic losses to poultry industries. Considering the high mutation rate and frequent genetic reassortment between segments in the genome of AIVs, emerging new strains are a real threat that may infect and spread through the human population, causing a pandemic. Therefore, rapid AIV diagnostic tests are essential tools for surveillance and assessing virus spreading. Real-time reverse transcription PCR (rRT-PCR), targeting the matrix gene, is the main official standard test for AIV detection, but the method requires well-equipped laboratories. Reverse transcription Loop-Mediated Isothermal Amplification (RT-LAMP) has been reported as a rapid method and an alternative to PCR in pathogen detection. The high mutation rate in the AIV genome increases the risk of false negative in nucleic acid amplification methods for detection, such as PCR and LAMP, due to possible mismatched priming. In this study, we analyzed 800 matrix gene sequences of newly isolated AIV in the EU and designed a highly efficient LAMP primer set that covers all AIV subtypes. The designed LAMP primer set was optimized in real-time RT-LAMP (rRT-LAMP) assay. The rRT-LAMP assay detected AIV samples belonging to nine various subtypes with the specificity and sensitivity comparable to the official standard rRT-PCR assay. Further, a two-color visual detection RT-LAMP assay protocol was adapted with the aim to develop on-site diagnostic tests. The on-site testing successfully detected spiked AIV in birds oropharyngeal and cloacal swabs samples at a concentration as low as 100.8 EID50 per reaction within 30 minutes including sample preparation. The results revealed a potential of this newly developed rRT-LAMP assay to detect AIV in complex samples using a simple heat treatment step without the need for RNA extraction.

Bloodstream infections and invasive nontyphoidal Salmonellosis in particular remain a major health and economic burden worldwide. The complexity of blood matrixes along with extremely low concentration of pathogens in blood poses a great challenge for rapid and ultrasensitive detection. Sample preparation has been the critical step that should provide blood-matrix-free sample with the targeted pathogen in the highest possible concentration. In this work, we addressed this challenge by combining magnetic-bead-based pathogen concentration and solid-phase PCR (SP-PCR). The SP-PCR performed on a supercritical angle fluorescence (SAF) microlens array embedded in a microchip enabled quick and accurate detection of low levels of Salmonella enterica serovar typhimurium and enteritidis in blood samples without culture enrichment. Protein AG-magnetic beads immobilized with antisalmonella antibody could efficiently concentrate both Salmonella serovars with a capturing efficiency >95%. Higher tolerance of Phusion hot start DNA polymerase to PCR inhibitors and its compatibility with protein AG-magnetic beads allowed the integration of SP-PCR. Analysis of Salmonella-spiked blood samples with the SP-PCR resulted in a limit of detection (LoD) as low as 86 CFU/mL and 94 CFU/mL for S. typhimurium and S. enteritidis, respectively, that could be attributed to the high fluorescence collection efficiency of the SAF microlens array. These combinations reduced the duration of analysis to less than 3 h including sample preparation. This platform has the potential for wide application as a high-throughput biosensor to analyze pathogens in clinical, food, and environmental samples.

Rapid detection of foodborne pathogens at an early stage is imperative for preventing the outbreak of foodborne diseases, known as serious threats to human health. Conventional bacterial culturing methods for foodborne pathogen detection are time consuming, laborious, and with poor pathogen diagnosis competences. This has prompted researchers to call the current status of detection approaches into question and leverage new technologies for superior pathogen sensing outcomes. Novel strategies mainly rely on incorporating all the steps from sample preparation to detection in miniaturized devices for online monitoring of pathogens with high accuracy and sensitivity in a time-saving and cost effective manner. Lab on chip is a blooming area in diagnosis, which exploits different mechanical and biological techniques to detect very low concentrations of pathogens in food samples. This is achieved through streamlining the sample handling and concentrating procedures, which will subsequently reduce human errors and enhance the accuracy of the sensing methods. Integration of sample preparation techniques into these devices can effectively minimize the impact of complex food matrix on pathogen diagnosis and improve the limit of detections. Integration of pathogen capturing bio-receptors on microfluidic devices is a crucial step, which can facilitate recognition abilities in harsh chemical and physical conditions, offering a great commercial benefit to the food-manufacturing sector. This article reviews recent advances in current state-of-the-art of sample preparation and concentration from food matrices with focus on bacterial capturing methods and sensing technologies, along with their advantages and limitations when integrated into microfluidic devices for online rapid detection of pathogens in foods and food production line.

Hepatic differentiation of human‐induced pluripotent stem cells (hiPSCs) under flow conditions in a 3D scaffold is expected to be a major step forward for construction of bioartificial livers. The aims of this study were to induce hepatic differentiation of hiPSCs under perfusion conditions and to perform functional comparisons with fresh human precision‐cut liver slices (hPCLS), an excellent benchmark for the human liver in vivo. The majority of the mRNA expression of CYP isoenzymes and transporters and the tested CYP activities, Phase II metabolism, and albumin, urea, and bile acid synthesis in the hiPSC‐derived cells reached values that overlap those of hPCLS, which indicates a higher degree of hepatic differentiation than observed until now. Differentiation under flow compared with static conditions had a strong inducing effect on Phase II metabolism and suppressed AFP expression but resulted in slightly lower activity of some of the Phase I metabolism enzymes. Gene expression data indicate that hiPSCs differentiated into both hepatic and biliary directions. In conclusion, the hiPSC differentiated under flow conditions towards hepatocytes express a wide spectrum of liver functions at levels comparable with hPCLS indicating excellent future perspectives for the development of a bioartificial liver system for toxicity testing or as liver support device for patients.

One of the fundamental steps needed to design functional tissues and, ultimately organs is the ability to fabricate thick and densely populated tissue constructs with controlled vasculature and microenvironment. To date, bioprinting methods have been employed to manufacture tissue constructs with open vasculature in a square-lattice geometry, where the majority lacks the ability to be directly perfused. Moreover, it appears to be difficult to fabricate vascular tissue constructs targeting the stiffness of soft tissues such as the liver. Here we present a method for the fabrication of thick (e.g. 1 cm) and densely populated (e.g. 10 million cells·mL−1) tissue constructs with a three-dimensional (3D) four arm branch network and stiffness in the range of soft tissues (1–10 kPa), which can be directly perfused on a fluidic platform for long time periods (>14 days). Specifically, we co-print a 3D four-arm branch using water-soluble Poly(vinyl alcohol) (PVA) as main material and Poly(lactic acid) (PLA) as the support structure. The PLA support structure was selectively removed, and the water soluble PVA structure was used for creating a 3D vascular network within a customized extracellular matrix (ECM) targeting the stiffness of the liver and with encapsulated hepatocellular carcinoma (HepG2) cells. These constructs were directly perfused with medium inducing the proliferation of HepG2 cells and the formation of spheroids. The highest spheroid density was obtained with perfusion, but overall the tissue construct displayed two distinct zones, one of rapid proliferation and one with almost no cell division and high cell death. The created model, therefore, simulate gradients in tissues of necrotic regions in tumors. This versatile method could represent a fundamental step in the fabrication of large functional and complex tissues and finally organs. Statement of Significance Vascularization within hydrogels with mechanical properties in the range of soft tissues remains a challenge. To date, bioprinting have been employed to manufacture tissue constructs with open vasculature in a square-lattice geometry that are most of the time not perfused. This study shows the creation of densely populated tissue constructs with a 3D four arm branch network and stiffness in the range of soft tissues, which can be directly perfused. The cells encapsulated within the construct showed proliferation as a function of the vasculature distance, and the control of the micro-environment induced the encapsulated cells to aggregate in spheroids in specific positions. This method could be used for modeling tumors and for fabricating more complex and densely populated tissue constructs with translational potential.

In this paper, we present, to the best of our knowledge, for the first time, in-depth theoretical analysis and experimental results for the optimisation of supercritical angle fluorescence (SAF) structures in polymer microfluidic chips fabricated from a combination of micro-milling and polymer injection-moulding techniques for their application in the highly-sensitive detection of pathogens. In particular, we address experimentally and theoretically the relationship between the supercritical angle and the heights of the SAF structures embedded in the microfluidic chips to obtain optimised results where the highest fluorescence intensity is collected, and hence determining the optimised limit of detection (LOD). Together with theoretical modelling, we experimentally fabricate microarrays of SAF structures with different heights varying from zero to the order of 300 μm in cyclic olefin copolymer (COC) microfluidic chips. The results show that for fluorophores at the interface of air and COC, the highest fluorescence intensities are obtained at SAF structures with a 163 μm height for a milling tool with a 97.4 μm diameter, which is in excellent agreement with our modelling. A fluorescence LOD of 5.42 × 104 molecules is achieved when using such SAF structures. The solid-phase polymerase chain reaction (SP-PCR) on these SAF structures permits sensitive pathogen detection (3.37 × 102 copies of the E. coli genome per μL) on-chip. These results especially are of interest for applications in hypersensitive pathogen detection as well as in assisting the design of devices for point-of-care applications. Findings on the height optimization of SAF structures also advance our understanding of SAF detection techniques and provide insights into the development of fluorescence microscopy.

Foodborne salmonellosis remains a major economic burden worldwide and particularly for food industries. The diverse and complexity of food matrices pose great challenges for rapid and ultra-sensitive detection of Salmonella in food samples. In this study, combination of pathogen pre-concentration with rapid molecular identification is presented to overcome these challenges. This combination enabled effective real-time PCR detection of low levels of Salmonella enterica serovar Typhimurium without culture enrichment. Anti-salmonella antibody, immobilized on protein AG-magnetic beads, could efficiently concentrate Salmonella Typhimurium with a capturing efficiency of 95%. In the direct PCR, a strong linear relationship between bacteria concentration and the number of cycles was observed with a relative PCR efficiency of ∼92% resulting in a limit of detection (LoD) of ∼2 CFU/mL. Analysis of spiked food samples that include vegetable salad, egg yolk, egg white, whole egg and minced pork meat has validated the precision of the method. A relative accuracy of 98.3% with a sensitivity of 91.6% and specificity of 100% was achieved in the Salmonella spiked food samples. The use of a Phusion hot start DNA polymerase with a high tolerance to possible PCR inhibitors allowed the integration of direct PCR, and thereby reducing the duration of analysis to less than 3 h. The Cohen's kappa index showed excellent agreement (0.88) signifying the capability of this method to overcome the food matrix effects in rapid and ultra-sensitive detection of Salmonella in food. This approach may lay a future platform for the integration into a Lab-on-a-chip system for online monitoring of foodborne pathogens.

Campylobacteriosis is one of the most common foodborne diseases worldwide. Two Campylobacter species – C. jejuni and C. coli in poultry and poultry products are considered to be the main source of human campylobacteriosis. Therefore, studying Campylobacter status in poultry flocks is needed to prevent transmission of disease and reduce human risk, health cost, and economic losses. In this study, we adapted and used a Loop-Mediated Isothermal Amplification (LAMP) assay for specific, sensitive, simple and cost-effective rapid detection of C. jejuni and C. coli in the poultry production chain. Amplified LAMP products were detected using a small, low-cost portable commercial blue LED transilluminator and a direct visual detection strategy was demonstrated. By using optimized conditions for amplification a limit of detection (LOD) of 50 CFU/ml was achieved for testing of C. jejuni and C. coli in spiked chicken feces without enrichment. The method took 60–70 min from receiving the samples to the final results (including 30 min for amplification). The optimized LAMP showed a relative accuracy of 98.4%, a specificity of 97.9%, and a sensitivity of 100% in comparison to real-time PCR method. Cohen’s kappa index also showed an excellent agreement (0.94) between the two methods. The results showed that the method is specific, sensitive and is suitable to develop for rapid detection of Campylobacter spp. at poultry production.

This paper provides new insights into the process of rapid, low-cost and large-scale fabrication of polymer microfluidic chips containing three-dimensional microstructures using in point of care devices, for applications such as detection of pathogens via a molecular diagnostic method, namely polymerase chain reaction (PCR). The details of fabrication methods are described. Do’s and don’ts are given to help both the experimentalist in labs and in industrial production to avoid doom fabrication issues.

This protocol provides insights into the rapid, low-cost, and largescale fabrication of polymer microfluidic chips containing three-dimensional microstructures used in point-of-care devices for applications such as detection of pathogens via molecular diagnostic methods. The details of the fabrication methods are described in this paper. This study offers suggestions for researchers and experimentalists, both at university laboratories and in industrial companies, to prevent doom fabrication issues. For a demonstration of bio-application in point-of-care testing, the 3D microarrays fabricated are then employed in multiplexed detection of Salmonella (Salmonella Typhimurium and Salmonella Enteritidis), based on a molecular detection technique called solid-phase polymerase chain reaction (SP-PCR).

LAMP has received great interest and is widely utilized in life sciences for nucleic acid analysis. To monitor a real-time LAMP assay, a fluorescence DNA dye is an indispensable component and therefore the selection of a suitable dye for real-time LAMP is a need. To aid this selection, we investigated the inhibition effects of twenty-three DNA dyes on real-time LAMP. Threshold time (Tt) values of each real-time LAMP were determined and used as an indicator of the inhibition effect. Based on the inhibition effects, the dyes were classified into four groups: (1) non-inhibition effect, (2) medium inhibition effect, (3) high inhibition effect, and (4) very high inhibition effect. The signal to noise ratio (SNR) and the limit of detection (LOD) of the dyes in groups 1, 2, and 3 were further investigated, and possible inhibition mechanisms of the DNA dyes on the real-time LAMP are suggested and discussed. Furthermore, a comparison of SYTO 9 in different LAMP reactions and different systems is presented. Of the 23 dyes tested, SYTO 9, SYTO 82, SYTO 16, SYTO 13, and Miami Yellow were the best dyes with no inhibitory effect, low LOD and high SNR in the real-time LAMP reactions. The present classification of the dyes will simplify the selection of fluorescence dye for real-time LAMP assays in point of care setting.

Concentration of pathogens directly from food samples by using magnetic beads is a potential strategy in the on-site food sample analysis. In this study, magnetic beads, double modified with platinum/palladium nanoparticle (Pt/Pd NP) and DNA aptamer, is presented as catalytic nano-bioprobes for the on-site detection of Salmonella enterica serovar Typhimurium in food and fecal samples. Combination of the developed catalytic nano-bioprobes with loop mediated isothermal amplification (LAMP) enabled rapid detection of low levels of S. Typhimurium in food and chicken fecal samples without culture enrichment. S. Typhimurium-specific DNA aptamer immobilized on the magnetic bead could efficiently concentrate S. Typhimurium with a capturing efficiency higher than 76% in phosphate-buffered saline (PBS). Further, DNA-mediated inhibition of peroxidase-mimic activity of Pt/Pd NP in combination with LAMP was used as a unique approach to detect S. Typhimurium. With this unique approach, it was possible to capture and detect S. Typhimurium as low as 10–15 CFU/mL in chicken meat sample and 3–10 CFU/mL in both whole egg and chicken fecal samples within less than 3 h. Analysis of S. Typhimurium-spiked chicken meat, whole egg and chicken fecal materials have confirmed the precision of the method. A relative accuracy of 90% with an intra and inter assay precision of 8.36% and 9.92% respectively was achieved in the S. Typhimurium-spiked food samples. This unique approach has the potential for the integration in to Lab-on-a-chip based biosensors for on-site monitoring of foodborne pathogens in future.

The genus Arcobacter (Vandamme et al., 1991), comprised of Campylobacter-related species, are considered zoonotic emergent pathogens. The presence of Arcobacter in food products like shellfish, has an elevated incidence worldwide. In this study, we developed a specific viable quantitative PCR (v-qPCR), using the dye propidium monoazide (PMA), for quantification of the viable Arcobacter spp. cells in raw oysters and mussels. The high selectivity of primers was demonstrated by using purified DNA from 38 different species, 20 of them from the genus Arcobacter. The optimization of PMA concentration showed that 20 μM was considered as an optimal concentration that inhibits the signal from dead cells at different concentrations (OD550 from 0.2 to 0.8) and at different ratios of live: dead cells (50:50 and 90:10). The v-qPCR results from shellfish samples were compared with those obtained in parallel using several culture isolation approaches (i.e., direct plating on marine and blood agar and by post-enrichment culturing in both media). The enrichment was performed in parallel in Arcobacter-CAT broth with and without adding NaCl. Additionally, the v-qPCR results were compared to those obtained with traditional quantitative (qPCR). The v-qPCR and the qPCR resulted in c.a. 94% of positive detection of Arcobacter vs. 41% obtained by culture approaches. When examining the reduction effect resulting from the use of v-qPCR, samples pre-enriched in Arcobacter-CAT broth supplemented with 2.5% NaCl showed a higher reduction (3.27 log copies) than that of samples obtained directly and those pre-enriched in Arcobacter-CAT broth isolation (1.05 and 1.04). When the v-qPCR was applied to detect arcobacter from real shellfish samples, 15/17 samples tested positive for viable Arcobacter with 3.41 to 8.70 log copies 1g-1. This study offers a new tool for Arcobacter surveillance in seafood.

Microcontrollers are programmable, integrated circuit chips. In the last two decades, their applications to industrial instruments, vehicles, and household appliances have reached the extent that microcontrollers are now the number-one selling electronic chip of all kinds. Simultaneously, the field of lab-on-a-chip research and technology has seen major technological leaps towards sample handling, sample preparation, and sensing for use in molecular diagnostic devices. Yet, the transformation from a laboratory based lab-on-a-chip technology to actual point-of-care device products has largely been limited to a fraction of the foreseen potential. We believe that increased knowledge of the vast possibilities that becomes available with open source microcontrollers, especially when embedded in easy-to-use development environments, such as the Arduino or Raspberry Pi, could potentially solve and even bridge the gap between lab-on-a-chip technology and real-life point of care applications. The profuse availability and extraordinary capabilities of microcontrollers, namely within computation, communication, and networking, combined with easy-to-use development environments, as well as a very active and fast moving community of makers, who are eager to share their knowledge, could potentially be the difference between a dreadful “chip-in-a-lab”-situation, and the next successful start-up. Here follows a brief insight into how open source microcontrollers could potentially have a transformative effect on the field of lab-on-a-chip research and technology. Details in some specific areas of application are briefly treated before addressing challenges and future perspectives.

We present an all-polymer, single-use microfluidic chip system produced by injection moulding and bonded by ultrasonic welding. Both techniques are compatible with low-cost industrial mass-production. The chip is produced for magnetic bead-based solid-phase extraction facilitated by immiscible phase filtration and features passive liquid filling and magnetic bead manipulation using an external magnet. In this work, we determine the system compatibility with various surfactants. Moreover, we quantify the volume of liquid co-transported with magnetic bead clusters from Milli-Q water or a lysis-binding buffer for nucleic acid extraction (0.1 (v/v)% Triton X-100 in 5 M guanidine hydrochloride). A linear relationship was found between the liquid carry-over and mass of magnetic beads used. Interestingly, similar average carry-overs of 1.74(8) nL/mg and 1.72(14) nL/mg were found for Milli-Q water and lysis-binding buffer, respectively.

Ultrasonic welding is a rapid, promising bonding method for the bonding of polymer chips; yet its use is still limited. We present two lab-on-a-chip applications where ultrasonic welding can be preferably applied: (1) self-aligned gapless bonding of a two-part chip with a tolerance of 50 μm; (2) bonding of a large area shallow chamber (1.8 cm(2) × 150 μm). Using injection moulding combined with ultrasonic welding we achieved a total production and bonding time of 60 s per chip, and a batch of chips could be produced within a day going from design to finished chips. We believe that the technical solutions offered here can significantly help bridge the gap between academia and industry, where the differences in production methods and materials pose a challenge when transferring technology.

A miniaturised array of supercritical angle fluorescence (SAF) micro-optics embedded in a microfluidic chamber was fabricated by injection moulding. The fabricated chip could enhance the fluorescence signal around 46 times compared to a conventional microscope. Collection of the fluorescence signal from the SAF array is almost independent of the numerical aperture, and the limit of detection was improved 36-fold using a simple and inexpensive optical detection system.

A method for fabricating a three-dimensional carbon structure (4) is disclosed. A mould (1) defining a three-dimensional shape is provided, and natural protein containing fibres are packed in the mould (1) at a predetermined packing density. The packed natural protein containing fibre structure (3) undergoes pyrolysis, either while still in the mould (1) or after having been removed from the mould (1). Thereby a three-dimensional porous and electrically conducting carbon structure (4) having a three-dimensional shape defined by the three-dimensional shape of the mould (1) and a porosity defined by the packing density of the packed natural protein containing fibre structure (3) is obtained. The carbon structure (4) is well suited for use as a scaffold for tissue engineering, or for material for batteries, fuel cells, supercapacitors, sorbents for separation processes, gas storage, supports for many important catalysts, etc.

Solid-phase PCR (SP-PCR) has attracted considerable interest in different research fields since it allows parallel DNA amplification on the surface of a solid substrate. However, the applications of SP-PCR have been hampered by the low efficiency of the solid-phase amplification. In order to increase the yield of the solid-phase amplification, we studied various parameters including the length, the density, as well as the annealing position of the solid support primer. A dramatic increase in the signal-to-noise (S/N) ratio was observed when increasing the length of solid support primers from 45 to 80 bp. The density of the primer on the surface was found to be important for the S/N ratio of the SP-PCR, and the optimal S/N was obtained with a density of 1.49 × 1011 molecules/mm2. In addition, the use of solid support primers with a short overhang at the 5' end would help improve the S/N ratio of the SP-PCR. With optimized conditions, SP-PCR can achieve amplification efficiency comparable to conventional PCR, with a limit of detection of 1.5 copies/μl (37.5 copies/reaction). These improvements will pave the way for wider applications of SP-PCR in various fields such as clinical diagnosis, high-throughput DNA sequencing, and single-nucleotide polymorphism analysis. Graphical abstract Schematic representation of solid-phase PCR.

A combined 2D microfluidic-microarray high throughput approach is reported to identify universal bacterial capturing ligands that can be tethered on the surface of 3D sponges fabricated by different methods for concentrating of bacterial targets in diagnosis devices. The developed platform allows for the first time the simultaneous monitoring of various ligands' affinities to different bacteria species in a dynamic condition in vitro. Moreover, it has been feasible to recognize the effect of steric hindrance on the function of capturing motifs through immobilizing spacer molecules with different lengths between the solid surface and ligands. 3D sponges and micropillars are modified with the most potent capturing molecule to assess their bacterial capturing in real blood samples. Next, the 3D structures are placed into a chip with an immense potential to recognize bacteria through imaging and fluorescence intensity concept.

Salmonellosis, an infectious disease caused by Salmonella spp., is one of the most common foodborne diseases. Isolation and identification of Salmonella by conventional bacterial culture method is time consuming. In response to the demand for rapid on line or at site detection of pathogens, in this study, we developed a multiplex Direct PCR method for rapid detection of different Salmonella serotypes directly from pork meat samples without any DNA purification steps. An inhibitor-resistant Phusion Pfu DNA polymerase was used to overcome PCR inhibition. Four pairs of primers including a pair of newly designed primers targeting Salmonella spp. at subtype level were incorporated in the multiplex Direct PCR. To maximize the efficiency of the Direct PCR, the ratio between sample and dilution buffer was optimized. The sensitivity and specificity of the multiplex Direct PCR were tested using naturally contaminated pork meat samples for detecting and subtyping of Salmonella spp. Conventional bacterial culture methods were used as reference to evaluate the performance of the multiplex Direct PCR. Relative accuracy, sensitivity and specificity of 98.8%; 97.6% and 100%, respectively, were achieved by the method. Application of the multiplex Direct PCR to detect Salmonella in pork meat at slaughter reduces the time of detection from 5 to 6 days by conventional bacterial culture and serotyping methods to 14 h (including 12 h enrichment time). Furthermore, the method poses a possibility of miniaturization and integration into a point-of-need Lab-on-a-chip system for rapid online pathogen detection.

Solid-phase PCR (SP-PCR) has become increasingly popular for molecular diagnosis and there have been a few attempts to incorporate SP-PCR into lab-on-a-chip (LOC) devices. However, their applicability for on-line diagnosis is hindered by the lack of sensitive and portable on-chip optical detection technology. In this paper, we addressed this challenge by combining the SP-PCR with super critical angle fluorescence (SAF) microlens array embedded in a microchip. We fabricated miniaturized SAF microlens array as part of a microfluidic chamber in thermoplastic material and performed multiplexed SP-PCR directly on top of the SAF microlens array. Attribute to the high fluorescence collection efficiency of the SAF microlens array, the SP-PCR assay on the LOC platform demonstrated a high sensitivity of 1.6 copies/µL, comparable to off-chip detection using conventional laser scanner. The combination of SP-PCR and SAF microlens array allows for on-chip highly sensitive and multiplexed pathogen detection with low-cost and compact optical components. The LOC platform would be widely used as a high-throughput biosensor to analyze food, clinical and environmental samples.

We present the application of electrochemical impedance spectroscopy (EIS) as a method for discriminating between different polydimethylsiloxane (PDMS) scaffolds for three-dimensional (3D) cell cultures. The validity of EIS characterisation for scaffolds having different degree of porosity (networks of structured or random channels) is discussed in relation to Archie’s law. Guidelines for EIS analysis are presented and demonstrated to provide porosity information in physiological buffer that agrees well with a more conventional weight-based approach. We also propose frequency ranges that may serve as means of single-frequency measurements for fast scaffold characterization combined with in vitro monitoring of 3D cell cultures.

The invention relates to a replication tool (1, 1a, 1b) for producing a part (4) with a microscale textured replica surface (5a, 5b, 5c, 5d). The replication tool (1, 1a, 1b) comprises a tool surface (2a, 2b) defining a general shape of the item. The tool surface (2a, 2b) comprises a microscale structured master surface (3a, 3b, 3c, 3d) having a lateral master pattern and a vertical master profile. The microscale structured master surface (3a, 3b, 3c, 3d) has been provided by localized pulsed laser treatment to generate microscale phase explosions. A method for producing a part with microscale energy directors on flange portions thereof uses the replication tool (1, 1a, 1b) to form an item (4) with a general shape as defined by the tool surface (2a, 2b). The formed item (4) comprises a microscale textured replica surface (5a, 5b, 5c, 5d) with a lateral arrangement of polydisperse microscale protrusions. The microscale protrusions may be provided on a flange portion of a first part and are configured to act as energy directors when forming an ultrasonic joint with a cooperating flange portion of a second part.

Scaffolds with multiple functionalities have attracted widespread attention in the field of tissue engineering due to their ability to control cell behavior through various cues, including mechanical, chemical, and electrical. Fabrication of such scaffolds from clinically approved materials is currently a huge challenge. The goal of this work was to fabricate a tissue engineering scaffold from clinically approved materials with the capability of delivering biomolecules and direct cell fate. We have used a simple 3D printing approach, that combines polymer casting with supercritical fluid technology to produce 3D interpenetrating polymer network (IPN) scaffold of silicone-poly(2-hydroxyethyl methacrylate)-co-poly(ethylene glycol) methyl ether acrylate (pHEMA-co-PEGMEA). The pHEMA-co-PEGMEA IPN materials were employed to support growth of human mesenchymal stem cells (hMSC), resulting in high cell viability and metabolic activity over a 3 weeks period. In addition, the IPN scaffolds support 3D tissue formation inside the porous scaffold with well spread cell morphology on the surface of the scaffold. As a proof of concept, sustained doxycycline (DOX) release from pHEMA-co-PEGMEA IPN was demonstrated and the biological activity of released drug from IPN was confirmed using a DOX regulated green fluorescent reporter (GFP) gene expression assay with HeLa cells. Given its unique mechanical and drug releasing characteristics, IPN scaffolds may be used for directing stem cell differentiation by releasing various chemicals from its hydrogel network.

The interferon-γ release assay (IGRA) is a widely used test for the presence of a cell-mediated immune (CMI) response in vitro. This measure is used to test for infection with intracellular pathogens or for validating vaccine efficacy, and it is a widely used test for both human as well as cattle. However, there is no consensus whether to use whole blood cultures or purified PBMCs for the assay, and both cell populations are being used and results compared. Therefore the aim of this study was to compare different culture settings using immune cells from previously vaccinated calves, and to shed light on external factors that could influence the read out in terms of IFN-γ levels. It was found that optimal culture conditions varied between individual animals; when polyclonal activated, cells from whole blood cultures were most responsive, but when activated specifically, the optimal cell concentration/population varied with whole blood, 10×10(6)cells/ml PBMC and 5×10(6)cells/ml PBMC being the highest performing conditions. A further investigation of the distribution of cell populations in PBMCs compared to whole blood was conducted, and a significant (p

In vitro generation of dendritic-like cells through differentiation of peripheral blood monocytes is typically done using two-dimensional polystyrene culture plates. In the process of optimising cell culture techniques, engineers have developed fluidic micro-devises usually manufactured in materials other than polystyrene and applying three-dimensional structures more similar to the in vivo environment. Polydimethylsiloxane (PDMS) is an often used polymer for lab-on-a-chip devices but not much is known about the effect of changing the culture surface material from polystyrene to PDMS. In the present study the differentiation of porcine monocytes to monocyte-derived dendritic cells (moDCs) was investigated using CD172apos pig blood monocytes stimulated with GM-CSF and IL-4. Monocytes were cultured on surfaces made of two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS and carbonised three-dimensional PDMS. Cells cultured conventionally (on two-dimensional polystyrene) differentiated into moDCs as expected. Interestingly, gene expression of a wide range of cytokines, chemokines, and pattern recognition receptors was influenced by culture surface material and architecture. Distinct clustering of cells, based on similar expression patterns of 46 genes of interest, was seen for cells isolated from two- and three-dimensional polystyrene as well as two- and three-dimensional PDMS. Changing the material from polystyrene to PDMS resulted in cells with expression patterns usually associated with macrophage expression (upregulation of CD163 and downregulation of CD1a, FLT3, LAMP3 and BATF3). However, this was purely based on gene expression level, and no functional assays were included in this study which would be necessary in order to classify the cells as being macrophages. When changing to three-dimensional culture the cells became increasingly activated in terms of IL6, IL8, IL10 and CCR5 gene expression. Further stimulation with LPS resulted in a slight increase in the expression of maturation markers (SLA-DRB1, CD86 and CD40) as well as cytokines (IL6, IL8, IL10 and IL23A) but the influence of the surfaces was unchanged. These findings highlights future challenges of combining and comparing data generated from microfluidic cell culture-devices made using alternative materials to data generated using conventional polystyrene plates used by most laboratories today.

Impedance is a promising technique for sensing the overall process of tissue engineering. Different electrode configurations can be used to characterize the scaffold that supports cell organization in terms of hydrogel polymerization and degree of porosity, monitoring cell loading, cell proliferation as well as the spatial distribution of cell aggregates in 3D. We have previously shown that impedance measurements allow accurate determination of conductivity in physiological solutions independent of validation and analysis of a specific equivalent circuit. Similarly to a physiological solution, cell culture medium conductivity, and hence the measured impedance, can respond to proliferating or dying cells populating the scaffold. Impedance may therefore be a key parameter for monitoring the biochemical dynamics that modulate 3D mammalian cell cultures over time. Furthermore, the conductivity of the medium filling the pores of the scaffold can serve as the basis for porosity determination using Archie’s law. Different networks of structured or random channels and degree of porosity can be detected. In addition, by combining a number of two-, three- and four-terminal (2T, 3T, 4T) configurations, it is possible to obtain complementary information on spatial distribution of cells in a 3D scaffold. 2T- and 3T configurations also reflect the impedance at the interface between an electrode and cell-loaded scaffold (polarization impedance, Zp), which may convey a further degree of information about the biochemical phenomena taking place in that sub-volume.

In this study, we explore the potential of electrical impedance tomography (EIT) for miniaturised 3D samples to provide a non-invasive approach for future applications in tissue engineering and 3D cell culturing. We evaluated two different electrode configurations using an array of nine circular chambers (Ø 10 mm), each having eight gold plated needle electrodes vertically integrated along the chamber perimeter. As first method, the adjacent electrode configuration was tested solving the computationally simple back-projection algorithm using Comsol Multiphysics in time-difference EIT (t-EIT). Subsequently, a more elaborate method based on the “polar-offset” configuration (having an additional electrode at the centre of the chamber) was evaluated using linear t-EIT and linear weighted frequency-difference EIT (f-EIT). Image reconstruction was done using a customised algorithm that has been previously validated for EIT imaging of neural activity. All the finite element simulations and impedance measurements on test objects leading to image reconstruction utilised an electrolyte having an ionic strength close to physiological solutions. The chosen number of electrodes and consequently number of electrode configurations aimed at maximising the quality of image reconstruction while minimising the number of required measurements. This is significant when designing a technique suitable for tissue engineering applications where time-based monitoring of cellular behaviour in 3D scaffolds is of interest. The performed tests indicated that the method based on the adjacent configuration in combination with the back-projection algorithm was only able to provide image reconstruction when using a test object having a higher conductivity than the background electrolyte. Due to limitations in the mesh quality, the reconstructed image had significant irregularities and the position was slightly shifted toward the perimeter of the chamber. On the other hand, the method based on the polar-offset configuration combined with the customised algorithm proved to be suitable for image reconstruction when using non-conductive and cell-based test objects (down to 1% of the measurement chamber volume), indicating its suitability for future tissue engineering applications with polymeric scaffolds.

Campylobacter jejuni is a major cause of infectious diarrhoea worldwide but relatively little is known about its ecology. In this study, we examined its interactions with Acanthamoeba castellanii, a protozoan suspected to serve as a reservoir for bacterial pathogens. We observed rapid degradation of intracellular C.jejuni in A.castellanii 5 h post gentamicin treatment at 25°C. Conversely, we found that A.castellanii promoted the extracellular growth of C.jejuni in co-cultures at 37°C in aerobic conditions. This growth-promoting effect did not require amoebae - bacteria contact. The growth rates observed with or without contact with amoeba were similar to those observed when C.jejuni was grown in microaerophilic conditions. Preconditioned media prepared with live or dead amoebae cultivated with or without C.jejuni did not promote the growth of C.jejuni in aerobic conditions. Interestingly, the dissolved oxygen levels of co-cultures with or without amoebae - bacteria contact were much lower than those observed with culture media or with C.jejuni alone incubated in aerobic conditions, and were comparable with levels obtained after 24 h of growth of C.jejuni under microaerophilic conditions. Our studies identified the depletion of dissolved oxygen by A.castellanii as the major contributor for the observed amoeba-mediated growth enhancement.

DNA microarrays have become one of the most powerful tools in the field of genomics and medical diagnosis. Recently, there has been increased interest in combining microfluidics with microarrays since this approach offers advantages in terms of portability, reduced analysis time, low consumption of reagents, and increased system integration. Polymers are widely used for microfluidic systems, but fabrication of microarrays on such materials often requires complicated chemical surface modifications, which hinders the integration of microarrays into microfluidic systems. In this paper, we demonstrate that simple UV irradiation can be used to directly immobilize poly(T)poly(C)-tagged DNA oligonucleotide probes on many different types of plastics without any surface modification. On average, five- and fourfold improvement in immobilization and hybridization efficiency have been achieved compared to surface-modified slides with aminated DNA probes. Moreover, the TC tag only costs 30% of the commonly used amino group modifications. Using this microarray fabrication technique, a portable cyclic olefin copolymer biochip containing eight individually addressable microfluidic channels was developed and used for rapid and parallel identification of Avian Influenza Virus by DNA hybridization. The one-step, cost-effective DNA-linking method on non-modified polymers significantly simplifies microarray fabrication procedures and permits great flexibility to plastic material selection, thus making it convenient to integrate microarrays into plastic microfluidic systems. Electronic supplementary material The online version of this article (doi:10.1007/s00216-011-5459-4) contains supplementary material, which is available to authorized users.

Endemic of avian influenza virus (AIV) in Asia and epizootics in some European regions have caused considerable public concern on a possible pandemic of AIV. A rapid method for virus detection and effective surveillance in wild avian, poultry production as well as in humans is required. In this article, a DNA microarray-based solid-phase polymerase chain reaction (PCR) approach has been developed for rapid detection of influenza virus type A and for simultaneous identification of pathogenic virus subtypes H5 and H7. This solid-phase RT-PCR method combined reverse-transcription amplification of RNA extract in the liquid phase with sequence-specific nested PCR on the solid phase. A simple ultraviolet cross-linking method was used to immobilize the DNA probes over an unmodified glass surface, which makes solid-phase PCR a convenient possibility for AIV screening. The testing of 33 avian fecal and tracheal swab specimens was completed in less than 2 h with 94% accuracy.