This talk presents a study of label-free and real-time monitoring of cellular attachment response through an integrated microfluidic device, which has a plasmonic gold layer coated on a glass substrate and configured in the form of large-scale-arrayed nano-holes as the core sensing element. The large-scale-arrayed nano-holes were fabricated by template-stripping method, and single cell trapping units were combined to isolate and place single cells above the nano-hole substrate. By measuring the extraordinary optical transmission (EOT) spectral shift caused by the periodic nano-holes, we are able to characterize the dynamic chemical-induced effects on living cells cultured on porous substrate. Chemicals were also used to demonstrate the capability of this chip to study cellular attachment response to external stimuli.

Microfluidics enables small volume, multiplexed experiments to be performed with minimal amount of reagents and high throughput testing and monitoring of cell metabolites. In order to monitor cell growth accurately in real-time, integrated sensors would require a non-consumptive and non-contact method to minimise analyte utilisation and cross-contamination. The holographic sensor works by an equilibrative mechanism, and relies on an optical method to observe changes in the cell environment. The sensor consists of a smart hydrogel embedded with analyte-sensitive ligands and Ag⁰ fringes. Based on Bragg's law, the periodic fringes reflect incident light of different wavelength at a particular angle depending on the Donnan potential of the gel. A change in analyte concentration shifts the Donnan potential and consequently expands or contracts the gel. This results in a corresponding colourimetric shift which makes the holographic sensor an integrated system of a sensitive recognition element, transducer and display. In this talk, the use of a miniaturised holographic pH sensor embedded in a microfluidic chip will be discussed. The growth of a lactic acid bacterium, Lactobacillus casei, which is commonly found in probiotic beverages, was monitored with the sensor by examining the corresponding fall in pH resulting from its homolactic fermentation.

Biofilm formation on synthetic membranes, i.e., membrane biofouling, is a major problem encountered in membrane processes for water and wastewater treatment. Quantification of biofouling is often conducted destructively and hence, results only reflect a snapshot of biofouling processes. This limitation is mainly due to the lack of tools that allow us to monitor dynamics of biofouling without the need of dissembling the membrane testing systems. In a recent study, we developed a novel multichannel fluidic membrane biofilm flow cell that enables non-destructive monitoring of biofouling dynamics using confocal laser scanning microscopy (CLSM). As a proof of concept, we used Green Fluorescent Protein (GFP)-tagged Shewanella oneidensis as a model organism and examined its biofilm development on forward osmosis membranes. The temporal profiles of quantitative biofouling parameters were obtained without disrupting the continuous operation of the membrane testing system. We also demonstrated that, combining with fluorescent staining techniques; the dynamics of biofouling by natural, un-tagged bacteria could also be monitored using CLSM without dissecting the membranes. The microfluidic flow cell developed in this study is a promising tool for non-destructive evaluation of antifouling properties of novel membranes.

Many studies reported that it is challenging to apply enhanced biological phosphorus removal (EBPR) process at high temperature. Glycogen accumulating organisms (GAOs) could easily gain their dominance over poly-phosphate accumulating organisms (PAOs) when the operating temperature was in the range of 25^oC to 30^oC. Moreover, almost all the studies about temperature effect indicated C and P turnover rates were lower at higher temperature than lower temperature. The EBPR culture enriched at lower temperature was employed in those studies. However, a few successful EBPR processes operated at high temperature have been reported recently while few study focused on the temperature effect on the EBPR culture enriched at higher temperature. Thus, we studied the temperature and carbon source effect on the EBPR culture enriched at higher temperature. The C and P turnover rates (i.e., P release rate, carbon uptake rate, PHA synthesis rate, PHA degradation rate and P uptake rate etc.) were higher at higher temperature than lower temperature with all carbon sources using the EBPR culture enriched at higher temperature. The PHA/P ratios were lower with all the carbon sources at 30^oC than 20^oC which indicated more phosphorus would be up taken consuming the same amount of PHA at 30^oC. The Gly/PHA ratios indicated the amount of glycogen synthesis consuming per mol PHA. The ratios were lower with all carbon sources at 30^oC than 20^oC. EBPR performance was better at higher temperature than lower temperature using the EBPR culture enriched at higher temperature. Moreover, EBPR performance was best with C2 in which PHB was the major component of PHA.

By integrating multifunctional microfluidic chip with digital image analysis, here we report a fast-speed method for automatically and accurately enumerating CTCs (Circulating Tumor Cells). By eliminating the deviation induced by different criteria of diverse examiner and off-chip procedures, as well as significantly reducing the time cost of CTC examination, this study provides a strategy to narrow the gap between the existing chip-based CTC enumeration technologies and the commonly accepted clinical examinations.

We develop a multifunctional microfluidic chip which consists of three functional segments: blood filtering, cell isolating and cell positioning. There are three micro-valves to control the cell flow between segments. Firstly, the diluted blood was labelled by immune-magnetic beads which recognize EpCAM and pumped in to the filtering segment. 1500 micro-pillars were designed to eliminate all blood impurities which may jam the microchannel or interfere the fluorescent identification of CTC. EpCAM+ cells were isolated in next segment. Since the cells were forced to flow in a thin layer, the chip exhibits better isolation efficiency than traditional tube-based isolation. Also, the segment is gradually widened. The flow speed, as well as the dynamic force applied on cells, is corresponding weakened. Therefore, cells labeled with different amount of magnetic-beads would be captured in different areas of the segment, avoiding cell aggregation. Then 6000 V-shape micro-structures, whose dimension were specially design to single cell capture, were used for efficiently positioning single cells in the third segment. Finally, the whole segment was fluorescently imaged using a microscopy with automatic stage. We developed a piece of unique software to analysis all images with the same standard. The microchip benefits the image processing as follows: 1) cells were localized in limited areas, remarkably reducing the calculation workload; 2) the fixed size of V-shaped structures provides a reference to algorithm for excluding cell debris and other fluorescent spots by size comparison.

The chip performances were verified on 20 breast cancer patients. It was demonstrated that the proposed chip provided not only accurate CTC numbers, but also useful information for molecular diagnosis.

Jellyfish stinging capsules known as nematocysts are explosive, natural-injection systems with high potential as a drug-delivery platform. These organelles consist of a capsule containing a highly folded thin needle-like tubule and a matrix highly concentrated with charged constituents that enable the tubule to fire and penetrate a target. Here, the nematocysts’ dielectric properties were experimentally investigated using dielectrophoretic and electrorotational spectra with best fits derived from theoretical models. The dielectric characterization adds to our understanding of the nematocysts’ structure and function and is necessary for dielectrophoretic isolation and manipulation of the populations. As expected, the effect of monovalent and divalent exchange cations resulted in higher inner conductivity for the NaCl treated capsules, in agreement with their relative higher osmotic pressure. In addition, an efficient dielectrophoretic isolation of different nematocyst sub-populations was demonstrated, paving the way to an understanding of nematocysts’ functional diversity and the development of an efficient drug delivery platform.

Skin in vitro assays that can replace animal models are in high demand in the skin-care and pharmaceutical industry for toxicology and drug delivery research. Besides being slow and expensive, animal models are not ideal for assessing effects in humans because of species differences, and raise ethical concerns. With the EU 2007 REACH regulation having drastically increased the number of chemicals that need to be evaluated for toxicity, and the EU 2013 ban on the use of animals in cosmetic product testing, there is an urgent need to develop broader and alternative profiling technologies for safety and efficacy skin testing methods.

For in vitro skin safety assays, organotypic skin cultures are tipically used. However, the reconstruction of these skin equivalents is costly, labor intensive, requires substantial cell culture expertise, and suffers from high variability and low barrier function. These 3D models are cultured in tissue culture inserts under static, inefficient, non-physiological conditions, and are not compatible with automated in vitro assays. Microscopy observations are difficult because of the excessive distance from the objective.

Similarly, in vitro efficacy assays require trained personnel, are labour intensive or low-throughput. Moreover, technologies like the Franz diffusion cells tipically used for skin permeation tests are based on the use of excised human skin, whose availability is limited and whose sourcing is regulated.

With the aim to overcome these limitations we integrated skin culture and in vitro testing into one system. We have developed a microfluidic platform that enables us to reconstruct skin equivalents of superior quality under in vivo-like perfusion, when compared to static cultures. The skin equivalent is cultured on a porous support membrane that separates two fluidic compartments. The microfluidic system can easily be converted from a tissue culture reactor to an in vitro analysis system by means of a set of interchangeable lid and insets. Flow of media through the microfluidic compartments provides a dynamic, continuous supply of nutrients and simultaneous removal of metabolic waste products similar to the role of blood vessels in native human skin.

Compared to organotypic skin reconstructed on traditional tissue culture inserts, the skin-on-chip equivalent showed a morphologically superior architecture and improved barrier properties. The epidermal layer is thicker and demonstrates a columnar, polarized basal keratinocytes. The skin-on-chip equivalent also shows more intense expression of involucrin, filaggrin and loricrin, as well as of basement membrane-related proteins laminin-5 and collagen IV, thus demonstrating a tighter anchoring of the epidermis to the dermis.

The use of thin and optically transparent plastic materials and the miniaturized design allow for real-time, non-invasive imaging. This system is scalable and suitable for automation of both the culture and the safety and efficacy experimental protocols. Exploring advanced miniaturization and high-throughput possibilities can minimize the cost of each replicate.

Unlike other skin-on-a-chip approaches, we reconstructed for the first time a full thickness organotypic skin equivalent directly in a microfluidic device made of thermoplastic materials and suitable for mass production.

     This paper presents a novel biosensor capable of continuously monitoring specific molecules (i.e., adenosine triphosphate - ATP) in the human serum by integrating the aptamer probes into a nanofluidic device. The advantage lies in its real-time signal regeneration of biosensor without the need of uploading the clean solutions for washing process since the ionic gate in the nanofluidic device could block or allow the target molecules flow through periodically.

     Ion concentration polarization (ICP) is a transport phenomenon which is observed when an electric field is applied across the nanofluidic channels, for instance formed by using a nanoporous Nafion membrane. Once the ICP process is stabilized, an ion enrichment zone and a depletion zone are established at the both sides of the channels. These two effects have been utilized for a wide range of applications such as desalination [1], pre-concentration of target analyte species [2][3][4]. ATP is an important biomolecule found in the living cells (intra/extracellular) and it relates directly to many physiological and pathophysiological events. Current techniques of secreted biomarker detections were mainly based on ion conductivity measurement, so the correlation between the signals and disease progress was limited. There were several binding assays based platforms developed before, but the contradiction between portability and functionality of signal regeneration remained. For example, aptamer assay was demonstrated for continuous real-time small molecules measurement [5], but the washing process was required by uploading a large amount of clean buffer solutions by professional operators for aptamer signal regeneration, which is highly demanded for applications in domestic health monitoring.

     In response, we have designed an aptamer based nanofluidic device for the real-time and continuous ATP detection in a small amount of human serum (100 µl for each cycle) (Figure 1). Our proposed nanofluidic device could allow the real-time monitoring individual patient’s conditions so the proper therapy can be offered on time. The working mechanism of aptamer probes was studied in detail (Figure 2, 3). Unprecedentedly, we found that there is a strong relationship between the binding affinity of ligand-receptor and the detaching force caused by the hydrodynamic flow. The critical energy to detach the ATP from ATP-aptamer complex was calculated as 1.86 x 10^-21 J and this energy can be used to settle the critical flowrate (i.e., 1 µl/min; kinetic energy E = 1.99 x 10^-21 J). We demonstrated that the device could produce the clean buffer solutions and endure five washing cycles to regenerate the aptamer without significant decrease of signal (Figure 4).

     In conclusion, by converting patient samples (i.e., human serum) to be clean solution for washing through applying electrical field to the device, the washing process could be approached without uploading buffer solutions. Accordingly, the dynamic signals of biomarkers could be measured in real time. With further miniaturization into a small wearable device for remote health condition monitoring, this nanofluidic device could improve current healthcare facilities significantly. The flexibility of this nanofluidic device could offer the possibility to integrate different receptors (i.e., aptamers) to monitor other secreted biomarkers in sweat, saliva.

Uric acid (UA) is the purine metabolic product and has a relationship with many clinical diseases, such as gout, kidney disease and heart disease that can result in the high UA in the blood [1–3]. Many medical investigations have indicated that the rise of blood serum UA can cause cardiovascular disease [4]. Therefore, the monitoring of UA in blood is critical for evaluating the therapy of gout patient in a long time. The current screening method for clinically measurement of the blood UA is the uricase enzymatic approaches. The working principle is based on that uricase oxidase (UOD) enzyme catalyzes blood UA decomposion into allantoin and then the difference in absorbance at λ=290nm of the enzyme catalyzed product has a linear relationship with UA concentration.  However, the optics-based spectroscopic method requires bulky equipment and complicates sample pre-processing procedures, which cannot meet the need of point of care test (POCT).  Electrochemical methods have been widely used in biomedical application because of its many merits, such as portability, low cost, easy integration, rapid analysis, one typical example-glucometer.

In this abstract, we demonstrate the world’s first medical smartphone as an electrochemical analyzer, which is incorporated with the enzymatic test strip for point of care characterization of UA in peripheral whole blood. A disposable electrochemical uric acid test strip was connected to the electrochemical module integrated with the smartphone through the specific interface-a slot around the edge of smartphone. A 3 μL human peripheral whole blood drop is applied on the strip for UA characterization and compared to the clinical biochemical analyzer with satisfactory agreement. The proposed medical smartphone provides a mobile screening electrochemical station for point of care test of many biochemical parameters of human blood under flexible spot, which is a promising technology for meeting the urgent need of the mobile health application.

“A schematic view of the device design is illustrated in Figure 1. The measured result by proposed device as compared with the conventional clinical biochemical analyzer is shown in Fig.2. The relative standard deviations for all concentrations of UA was between 1.58% and 4.56% which are acceptable values for reproducibility in these device. Table I shows that UA concentrations measured by the medical smartphone reader were consistent with those by the commercial biochemical analyzer and that there was no significant difference among the three groups of UA test strips when measuring the same samples. These results indicates that the medical smartphone reader is highly reproducible and accurate for blood UA measurement suggesting a great potential for clinical use.

A Biosensor Combining Molecularly Imprinted Polymers (M-MIPs) and Surface Enhanced Raman Spectroscopy (SERS) to Detect Antibiotics in Food Samples

Yi Sun^,1*, Jon Ashley¹, Kaiyu Wu¹, and Anja Bosen¹.

¹ Department of Micro- and Nanotechnology, Technical University of Denmark, Ørsteds Plads, DK-2800 Kgs. Lyngby, Denmark

* Email: sun.yi@nanotech.dtu.dk; Tel.: +45 45256319

In this study, temperature-responsive magnetic molecularly imprinted polymers (M-MIP) nanoparticles were synthesized for the first time for the extraction of cloxacillin in pork products. By combining the M-MIPs with surface enhanced Raman spectroscopy (SERS), a sensitive biosensor was demonstrated to detect cloxacillian with pico-mole sensitivity.

MIPs are synthetic ligands which can be tailored to bind any analyte of choice¹.  They are of great interest due to their thermal stability, robustness, low cost and comparable binding affinity. They have been used in sample preparation and biosensing as an attractive alternative to natural antibodies to capture targets ranging from small molecules to big proteins.  

In this work, the magnetic nanoparticles with MIP-based receptors were synthesized for efficient and rapid extraction of antibiotic residues in pork samples.  Fe₃O₄ nanoparticles were obtained using the solvothermal synthesis.  The resultant nanoparticles were treated with Tetraethyl orthosilicate (TEOS) to form a SiO₂ layer.  Finally a thin MIP layer was polymerized round the nanoparticles using azobisisobutyronitrile (AIBN) as the initiator, ethylene glycol dimethacrylate (EDGMA) as the cross-linker, N-isopropylmethacryamide (NIPAm), methacrylic acid (MAA) as the monomers and the antibiotic as the template.  By adding the monomer NIPAm, the MIPs become temperature responsive, and can swell at low temperature to release the target. The corresponding magnetic non-imprinted polymer nanoparticles (M-NIP) was prepared using the same method in the absence of the template. An Overview of the synthesis strategy is shown in Fig. 1. The resultant M-MIP nanoparticles were characterized using IR, XRD scanning electron microscopy (SEM) and transmission electron microscopy (TEM) (Fig. 2).  Both binding affinities of the resultant M-MIPs and M-NIPs were tested using UV absorbance (Fig. 3). M-MIPs with 300-400 nm in size and good binding capacities were obtained.

To demonstrate the feasibility of using M-MIPs for sample preparation, the synthesized M-MIPs were mixed with pork blood samples spiked with Chloxacillian. After incubation at room temperature, the M-MIPs were collected using a magnet and washed by acetonitrile. Owing to the thermos-responsive properties of MIPs, Chloxacillian was easily released by cooling the MIPs to 4 degree. The collected Chloxacillian was dropped on a SERS substrate which contained an array of silicon micropillars coated with silver. The corresponding calibration plots showed a detection limit (LOD) of about 50 pmol (Fig. 4). The biosensor combining M-MIPs and SERS would be widely used on site or in the field for rapidly screening food contaminants to ensure food safety.

Fig. 1: Overview of the synthesis of M-MIPs

Fig.2 IR characterization of Fe₃O₄, Fe₃O₄@SiO₂, Fe₃O₄@ SiO₂-MPA and, Fe₃O₄@SiO₂-MIP; XRD of Fe₃O_4.

Fig.3 (A) Binding kinetics and (B) Binding capacity of Cloxacillian MIPs and NIPs.

Fig.4 SERS spectra of cloxacillin in MeOH:acetic acid (9:1) and corresponding calibration plots.

REFERENCES:

1. J. Ashley, M-A. Shahbazi, K. Kant, V. A. Chidambara, A.Wolff, D. D. Bang, Y. Sun, “Molecularly Imprinted Polymers for Sample Preparation and Biosensing in Food analysis: Progress and Perspectives, Biosens. Bioelectron. 2017, 91, 606-615.
2.