An automated real-time system for the rapid detection of protozoa, Giardia lamblia and Cryptosporidium, in drinking water is implemented based on the micro opto-fluidic system and image classification. Processing around 15 images of protozoa per second are realized in this system.

A single-measurement sweep-free distributed Brillouin optical time domain analyzer (BOTDA) sensor is proposed and experimentally demonstrated employing digital optical frequency comb (DOFC) probe signal for coherent detection of Brillouin Phase Spectrum (BPS), without any frequency scanning and data averaging. The phase shift of each frequency tone induced by Brillouin interaction is simultaneously measured in a single data acquisition.

We have developed a photometry system for cell number in suspensions of tissue culture cells. Cell suspensions are turbid and absorb and scatter the light. The higher the cell concentration, the higher the turbidity. Although spectrophotometers can measure intensity of light very accurately, this method is not widely used because of its large volume of sample requirement and bulky system. By using a led emitting diode as light source and photodiode as detector, we minimized the device to handheld size which can measure the optical density of cell suspension within the tissue culture hood. An optical measurement tip was designed, which only requires less than 50 µl. A standard curve of absorbance vs. cell density is used to estimate cell number with accuracy and reproducibility could compete with hemocytometer counting and with speed and ease surpassing use of a Coulter counter. The limit of detection of our system can reach 10,000 cell/mL.  The device should be readily extended to assays of cell number directly within microplate culture wells. The photometry assay described here is of significant use in all experiments requiring rapid, measurements of cell number, including determinations of cell doubling time and equal plating of parallel cultures.

Noble-metal nano-hole arrays that generate EOT phenomenon are a promising label-free surface plasmon resonance (SPR) biochemical sensor [1]. Cellular response detection by other SPR sensors (Kretschmann prism and photonic crystal) has been successful [2]. However, limited by the high cost of earlier nano-hole fabrication techniques such as FIB/EBL, the EOT-based biochemical sensors had only ~10x10 μm scale of nano-holes and thus were applied mostly in detecting molecules [1], which need only a small area of sensing elements. Recently, a template-stripping technique to fabricate large area (~1x1 cm) of nano-holes was reported [3], which enables EOT-based measurement of cellular events. Prior studies [4] showed that the cell adhesion on the nano-holes can result in a change in effective refractive index (RI), which, in turn, leads to a spectral shift in the transmission peak. Therefore, we hypothesize that the EOT-based sensors are capable of label-free monitoring the cellular effects of toxic chemicals that change cell adhesion conditions.

The integrated microfluidic device and measuring system shown in Fig. 1 was used to monitor the cellular response quantified by the EOT spectral shift in association with chemical stimuli. Gold film perforated with nano-hole arrays on a glass substrate was fabricated by depositing 100-nm-thick gold on a nano-hole-patterned silicon template and template-stripped onto the glass slide with UV curable epoxy [3]. PDMS microfluidic channels with an inlet and an outlet were fabricated by soft lithography and align-bonded with a gold-film-coated glass slide. After washing and sterilization of the device, cells were cultured on the cystimine-modified gold surface till confluent. Then the device was placed under a microscope connected to a spectrometer to record the spectral response (Fig. 2) to chemical stimuli applied via the microfluidic channels. Using the spectrum at t=0 as baseline, the spectral shift at different times was acquired as a function of the chemical concentration. In addition to chemical stimuli reported here, the same system can be used to characterize the cell response to electrical stimuli in the future.

We applied three types of chemical stimuli to HEK293 cells in experiments. The first was trypsin that breaks the collagen links between cells and the substrate. Fig. 3 shows the spectral shift for 0.05% trypsin in PBS. The spectral shift has a logarithmic time dependence, which fits well with the first order reaction mode of enzyme. The spectrum was blue-shifted because the effective RI near gold surface decreased as the cell were gradually detached from the substrate. The second stimulus was lipopolysaccharide (LPS), which is a cell toxic chemical known to flatten and expand cells. Fig. 4 plots the spectral shift for different concentrations of LPS, showing a red-shifted spectrum with increased time and concentration, which was attributed to a greater RI near the sensor surface due to increased cell adhesion on the gold nano-holes. The third stimulus was sodium azide, which, in contrast to LPS, makes cells to transform and shrink. As expected, the spectrum in Fig. 5 was blue-shifted, since the effective RI dropped in response to weaker cell adhesion. These results demonstrate the capability of our EOT-based microfluidic biosensor for label-free monitoring of cellular responses to external stimuli.

We demonstrated a fiber integrated optofluidic platform to perform miRNA detection with high sensitivity and specificity based on FRET principle. Recently miRNA has attracted extensive interest as a biomarker, providing important evidence for early disease diagnosis and post treatment. Traditionally, numerous analytical approaches such as northern blotting, micro arrays , and real-time quantitative polymerase chain reaction have been developed for miRNA detection. However, the existing methods have many technical challenges, for example, the concentration of miRNA in vivo is normally at low level, and the short sequence and easy degradation by RNAase makes it difficult for PCR. Therefore, we developed an innovative optofluidic platform that combines the advantage of droplet microfluidics and optofluidic technology, and provides a low-cost, compact and high specificity approach for identification and quantification of miRNA using FRET principle. Single-nucleotide differences within miRNA family can be distinguished by a novel Y-junction design of donor, receptor and miRNA sequence. We performed one step miRNA detection with simple device operation, and our platform achieved target miRNA identification at a low concentration and high specificity.

Aptamers can specifically bind to proteins, small molecules and cell receptors, which are considered as chemical antibodies. In this study, we present an aptamer-based magnetic nanoparticle for the quantitative and qualitative detection of Mucin-1, which is one kind of tumor biomarkers and is overexpressed in breast cancer.

Compared with antibody, aptamers display an outstanding nature of high target specificity and affinity, biocompatibility and stability, as well as no immunogenicity and low production cost. The changes of sizes and zeta potentials are used to monitor the synthesis process during the fabrication of MBs and probes. The structure of the SERS nanoprobe is core-shell type. Raman reporter molecules are modified in the middle of the core-shell structure. This structure can prevent the aggregation of nanoparticles and maintain the stability of the nanoprobe, as well as significantly enhance SERS signals (see Figure 1). Compared with Au NPs, the color of Au@Ag probe is change from red to yellowish as determined from the inset in Figure 2. It is obvious that a blue-shift of the extinction peak was observed when introducing a silver shell onto the core nanoparticle (see Figure 2). The thickness of the silver shell of the nanoprobe is approximately 1nm-3nm as determined from the TEM images (see Figure 3).

In this strategy, core-shell nanoparticle is used as the reporting probe and MB is utilized as the capturing substrate. In the absence of Mucin-1, the SERS nanoprobes are assembled around the MB due to DNA hybridization. The SERS intensity is negatively correlated with the concentration of target Mucin 1. With high specificity and sensitivity, this method would have a promising application in early-stage cancer diagnosis.

David Chauvin¹, Isabelle Ledoux-Rak¹ and Chi Thanh Nguyen^1,*

¹Laboratoire de Photonique Quantique et Moléculaire, UMR 8537, Ecole Normale Supérieure Paris-Saclay, CentraleSupélec, CNRS, Université Paris-Saclay, 61 avenue du Président Wilson 94235 Cachan, France

* Email: ctnguyen@lpqm.ens-cachan.fr; Tel.: +33-147405557

We report in this paper a new design and realization of a differential label-free optofluidic sensor based on polymeric vertically coupled microresonators for biochemical sensing. Label-free sensing based on optical polymeric microresonators has shown a very high performance in terms of detection limit [1]. When integrated into a microfluidic circuit to form an optofluidic device, it can provide an efficient and accuracy real-time monitoring of surface sensing and chemical reaction kinetics detection [2, 3]. But when using a single transducer configuration, the transduction signal of an optofluidic sensor is perturbated by various phenomena such as thermal drift, spikes of microfluidic pressure, mechanical vibration parasites, intensity variation of optical source. These perturbations severely reduce the accuracy of sensing and then the sensor detection limit. Differential measurement permits to overcome these perturbations and therefore to improve sensor performances. The differential measurement principle is based on a simultaneous comparison of an analyte transduction signal with a reference transduction signal measured under the same physical conditions. If these two signals are submitted to the same external perturbations, we can extract the net sensing signal by subtracting the analyte transduction signal from the reference signal. To assure that these two signals are in the same experimental conditions, the design, the fabrication and the set-up of the optofluidic sensor should be optimized.

A schematic view of the device is illustrated in Figure 1, and the concatenation of microscope photographs of the optical integrated device is presented in Figure 2. This device, composed of one Y-branch directional coupler and two identical vertically coupled microracetracks, was made of polymers (SU-8 photoresist and Cytop fluorocopolymer) deposited onto a silica lower cladding covering a silicon substrate. Microfluidic channels were fabricated with another polymer (PDMS) and covered the optical integrated circuit by applying a pressure via a specific mechanical set-up. The microfluidic flows injected into two sensor channels were provided by a microfluidic station (pump and two electrovalves). The optical detection setup was realized with two separated identical photodetectors. The optical source was a tunable laser emitting at 1500-1640 nm wavelengths.

The differential optofluidic sensor has demonstrated a real-time automatic correction of the long-term thermal drift and also of the short-term external perturbations in its response. Figure 3 presents the results of a homogeneous detection of glucose solution (27.9 mM glucose in deionized water) by the sensor. On the left, we observed a thermal drift on the transduction signals extracted from measurement and reference microracetracks and on the right, the real-time differential signal. In Figure 4 are presented the result of an automatic correction of short-term interferences (spikes on the signals) on the sensor response. We can apply also this sensing scheme for the surface detection in order to overcome not only external perturbations but also some non-specific effects of surface detection. A new multiplex sensing using our optofluidic sensor for surface detection of the couple streptavidin-biotin is going to be realized.

Fig.1 Schematic view of the differential label-free optofluidic sensor based on polymeric microracetracks.

Fig. 2 Concatenation of photographs under microscope of the polymer-based microracetracks device.

Fig. 3 Optical response of the sensor with thermal drift (left) and its differential response (right).

Fig. 4 External perturbations on the transduction signals of the sensor (left) and of its differential response (right).

REFERENCES:

[1] Delezoide, M. Salsac, J. Lautru, H. Leh, C. Nogues, J. Zyss, M. Buckle, I. Ledoux-Rak, and C. T. Nguyen, "Vertically coupled polymer microracetrack resonators for label-free biochemical sensors", Phot. Technol. Lett., 2012, 24, 270-272.

[2] Delezoide, J. Lautru, J. Zyss, I. Ledoux-Rak, and C. T. Nguyen, "Vertically coupled polymer microresonators for optofluidic label-free biosensors", Proc. of SPIE, 2012, 8264, 826416-13.

[]3 Chauvin, I. Ledoux-Rak and C.T. Nguyen, "Optimizing detection limits of optical resonator based sensors by optimization of real-time measurement of resonators response", Proc. of SPIE, 2016, 9899, 98991J.

Droplet digital PCR (ddPCR) is a revolutionary alternative to conventional real-time PCR for measuring nucleic acids with high precision and specificity, and is playing a more and more important role in biomedical research and clinical diagnostics. However, current commercial-available ddPCR systems requires different instruments to perform droplet generation and droplet reading, which makes the operation complicated and limited the wide application of ddPCR. To simplify the process, novel microfluidic devices have been reported, such as Abraham Lee’s 1-million droplet array device¹, centrifugal step emulsification chip², and SlipChip³. However, it’s still challenging to further simplify and reduce the cost of ddPCR for point-of-care applications.

In this work, we present a simple step emulsification ddPCR (SE-ddPCR) device which integrates sample loading, droplet generation, thermal cycling, and on-chip fluorescence imaging. Compared with previous methods, our compact device (1 inch × 3 inch in size) can simultaneously analyze 8 samples, generating around 10,000 droplets for each sample. The device with droplet array generated is shown in Fig 1. We designed a sample loading channel in the middle, and droplets were generated through nozzles based on step emulsification. The droplets arranged into monolayer array in the chamber automatically. This ddPCR device use a single pressure pump to drive droplet generation of 8 samples in parallel. Importantly, we used an optimized mineral oil instead of fluorinated oil, which further reduced the cost and avoided bubbling during thermal cycling².

We evaluated the performance of SE-ddPCR by measuring the concentration of a constructed plasmid containing HER2, a gene related to breast cancer. As shown in Fig 2, a linear regression was obtained in the range of 10 to 2000 DNA copies/μl, demonstrating high robustness of SE-ddPCR. Next, we measured copy number variation of HER2 in 16 clinical samples from patients with breast cancer, by simultaneous counting the HER2 gene and a reference gene (RNase P). The results of SE-ddPCR was compared with that using commercial QuantStudio 3D digital PCR system. Our SE-ddPCR system successfully detected the overexpression of HER2 in 10 samples, and the other 6 were identified as normal. The results were comparable with those obtained from the commercial systems. Current work is directed toward development of a fully automated SE-ddPCR system, which integrates liquid handling, SE-ddPCR and fluorescence imaging and analysis.

In this presentation, optical molecular detection techniques based on light localization are explored for optofluidic applications. For improved sensor characteristics in surface plasmon resonance (SPR) detection, surface-enhanced nanoarray structures have been investigated to create locally amplified electromagnetic near-fields on metallic substrates. Surface-enhanced plasmonic nanoarrays structures can create locally amplified electromagnetic near-fields as a consequence of evanescent field localization on metallic substrates. While the effect of light localization in the near-field using plasmonic nanoarrays may be moderate, the approach can be powerful when localized light fields are spatially colocalized with target molecular distribution. As such, various approaches to produce field matter colocalization for applications in detecting molecular interactions are to be discussed, for example, by oblique evaporation-based device fabrication and plasmonic lithography [1-5].

On the other hand, colocalization can be extended on a broader scale to general biomolecular detection beyond SPR and applied to microscopy and imaging. The creation of localized fields has been investigated in many studies in the past because of the potential to improve resolving power for imaging molecular processes typically impossible to observe under diffraction limit. Although emerging approaches have been extremely successful to produce super-resolved images, we explore alternative techniques based on plasmonic nanoarrays by which achievable resolution may be customized to fit the specific imaging needs and at the same time a conventional optical system may be used. Feasibility studies performed on visualizing internalization of virus particles [6], sliding microtubules and bacterial motility on random and periodic nanopatterns will be discussed [7-11]. Enhancement of axial resolution for the detection of intracellular protein distribution is also reported by extraordinary light transmission using graded plasmonic nanoapertures.

 It is also imperative to understand the force acting on molecules under detection for sensor and imaging applications. Figure 1a-c shows the gradient force produced by a plasmonic nanopost of diameter f = 100, 150, and 200 nm (height: 30 nm, gap between posts: 100 nm, and period: 750 nm) and clearly confirms the trapping force exerted at the post rim. Spectra of electric near-field maximum are also presented in Figure 1d (at the wavelength at which a maximum field strength is obtained for each nanopost, the gradient force in Fig. 1a-c was calculated). The results suggest the possibility of wavelength-dependent switching of the trapping force. This is only one of many application examples of the trapping force that can be applied and has thus been pursued in various optofluidic systems.

This paper reports the preparation of SERS substrate in microfluidic channels by electrostatic assembly, and quantitative detection of dopamine for high sensitivity and uniformity was achieved by using of the microfluidic-SERS system .

A schematic view of the microfluidic-SERS system is illustrated in Figure 1. Firstly, silver nanostars were prepared by reducing silver nitrate with hydroxylamine and sodium citrate^[1], and the TEM image is shown in figure 2. Then stable and sensitivity SERS substrate was successfully prepared in microfluidic chips by the electrostatic interaction between positively charged PDDA and negatively charged Ag nanostars. 4-MBA was selected as a raman reporter to test the sensitivity and reproducibility of the substrate.

Dopamine is a kind of important neurotransmitters in the body, which is able to adjust the mood of individuals, and it is proved that diseases such as Parkinson's disease and depression is associated with dopamine levels in the human body^[2,3]. Understand the exact dopamine concentrations in the human body can help the treatment of diseases such as Parkinson's disease. using the prepared microfluidic-SERS system described above, we have successful achieved label-free detection of  dopamine. Utilizing 4-MBA as raman reporter, Aptamer-based dopamine qualification was also applied in this system, figure 3 shows the result which exhibit high sensitivity and wide linear range from picomolar to micromolar concentration.