On-Chip Photothermal Analyte Detection Using Integrated

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On-chip photothermal analyte detection using integrated luminescent temperature sensors Simon Anton Pfeiffer, and Stefan Nagl Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02220 • Publication Date (Web): 28 Jul 2017 Downloaded from http://pubs.acs.org on July 31, 2017

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Analytical Chemistry

On-chip photothermal analyte detection using integrated luminescent temperature sensors Simon A. Pfeiffera and Stefan Nagla,b,* a Institut für Analytische Chemie, Universität Leipzig, Johannisallee 29, 04103 Leipzig, Germany b The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China ABSTRACT: Optical absorbance detection based on attenuated light transmission is limited in sensitivity due to short path lengths in microfluidic and other miniaturized platforms. An alternative is detection using the photothermal effect. Herein we introduce a new kind of photothermal absorbance measurement using integrated luminescent temperature sensor spots inside microfluidic channels. The temperature sensors were photopolymerized inside the channels from NOA 81 UV-curable thiolene prepolymer doped with tris(1,10-phenanthroline)ruthenium(II) temperature probe. The polymerized sensing structures were as small as 26 ± 3 µm in diameter and displayed a temperature resolution of better than 0.3 K between 20 and 50 °C. The absorbance from 532 nm laser excitation of the food dye Amaranth as a model analyte was quantified using these spots and the influence of the flow rate, laser power and concentration investigated. Calibration yielded a linear relationship between analyte concentration and the temperature signal in the channels. The limit of detection for the azo-dye Amaranth (E123) in this setup was 13 µM. A minimal detectable absorbance of 3.2*10-3 AU was obtained using an optical path length of 125 µm in this initial study. A microreactor with integrated temperature sensors was then employed for an absorbance-based miniaturized nitrite analysis yielding a detection limit of 26 µM at a total assay time of only 75 s. This technique is very promising for sensitive, and potentially capable of spatially-resolved, optical absorbance detection on the micro- and nanoscale.

The introduction and development of microfluidic “lab on a chip” systems has enabled miniaturization of many analytical procedures onto unprecedented scales, often very rapidly, and needing only minute sample amounts.1-4 However many routine analytical methods are still carried out on much larger scales. One issue that keeps microfluidics from being adopted more widely is the lack of easily accessible and applicable detection methods.4,5 At-line chemical analysis for many microfluidic platforms is enabled by techniques such as liquid chromatography-mass spectrometry (LC-MS) and gas chromatography mass spectrometry (GC-MS). Although these techniques are characterized by high sensitivity and analyte specificity they are restricted to fractions taken at certain outlets of the microfluidic network and are also limited by the analysis speed and costs. Online and inline methods, on the other hand allow, in principle, very fast readout at every position of a microfluidic chip. Therefore they provide information that cannot be obtained with at-line or offline methods and feature the potential for real-time continuous process control. The leading detection method in this area is by far fluorescence, but it usually requires at least one additional labeling step, and it is not applicable to many compounds. On a larger scale, many analytical parameters can be conveniently monitored using optical absorbance at a particular wavelength in the UV or visible spectral region. Using different wavelengths, the technique is relatively

universal, as almost all compounds of interest absorb somewhere in the UV/Vis range. However, the most common and easiest way to quantify absorbance is the monitoring of attenuated transmission, which is insensitive in miniaturized systems because the Beer-LambertBouguer law governs it. The latter establishes a linear relationship between the attenuation coefficient, the optical path length, and the species concentration, respectively, and the measured absorbance. To circumvent this limitation a variety of techniques for elongation of the optical path in microfluidic systems using particular channel geometries and optimized light coupling have been developed.6-11 An alternative absorbance detection modality, not limited by optical path length, is based on the photothermal effect. When a molecule is excited by absorption of a photon and subsequently undergoes non-radiative relaxation to ground state, the excess energy is dissipated to the surrounding medium and leads to local heating of the solution. A great variety of techniques exist to quantify the photothermal effect, which are subsumed under the term photothermal spectroscopy.12 Temperature affects many physical properties like refractive index and viscosity of the surrounding medium. Photothermal calorimetry is widely employed in macroscopic systems.13,14 One very common modality is photoacoustic detection or spectroscopy. A sinusoidally modulated (laser) light source is used, which in turn creates

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modulated thermal expansion leading to a pressure (or sound) wave that may be measured by a microphone.15 The technique is very sensitive and versatile, particularly for measurement on or in biological structures, but is poorly suited for microfluidic systems as they are enclosed in solid plates, resulting in strong attenuation of the signal. In microfluidics, the predominant photothermal absorbance detection technique is thermal lens spectroscopy (TLS). In TLS two lasers are employed, one pump laser to induce photoexcitation, and a second probe beam to detect changes in refractive index due to heating of the sample. Thermal lens spectroscopy on a microscope platform (TLM) has been implemented first by the Kitamori group and extensively used to detect absorbance in microfluidic devices.16 More recently the same group applied an interferometric technique for absorbance detection in extended nanochannels.17 In a related approach Schimpf et al. demonstrated photothermal analyte detection using a Young interferometer on a glass substrate.18 Dennis et al. used the temperature dependence of conductivity for photothermal detection in a multielectrode setup with laser excitation in a microchip electrophoresis system.19 Photothermal heating and quantification of the temperature increase using fluorescent temperature probes in aqueous solution was previously demonstrated in microfluidic systems.20,21 But in these works the photothermal effect was merely used as means of contactless heating and not for quantitative detection. Free probes that are not stationary and not shielded from the environment are not well suited for that. For integration of stationary luminescent chemical sensor layers into microfluidic chips, numerous ways have been devised in recent years.22-24 Temperature sensors had been introduced into glass and elastomer hybrid microfluidic systems by staining the elastomer with fluorescent probes,25 blade26 or spin coating27 of glass substrates, and subsequent sealing of the elastomer to the glass. Herein we explore photothermal absorbance detection in microfluidic chips using luminescent temperature sensor spots. Our recently developed technique for maskless photopolymerization of luminescent pH and oxygen sensors with diameters smaller than 50 µm inside glass microfluidic devices28 was adapted to create small temperature sensing spots inside microfluidic flow reactors. An inverted microscopic setup with excitation from top and bottom side was developed to couple an excitation laser and an LED for optical interrogation of temperature sensors into the microfluidic setup. The photothermal signal was characterized with respect to flow rate, laser power, and analyte concentration. The system was then applied to monitor nitrite concentration via a chromogenic reaction (the synthesis of an azo dye) within a micro flow reactor.

EXPERIMENTAL SECTION MATERIALS. Oligo ethylene glycol diacrylate (OEG-DA, average MW 258 Da), 3-(trimethoxysilyl)propyl methacrylate (TPM), 2,2-dimethoxy-2-phenylacetophenone

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(DMPA), sulfanilamide, N-(1-napthyl)ethylene diamine dihydrochloride, sodium nitrite, Amaranth (analytical standard), ruthenium(II)-tris(l,l0-phenanthroline) dichloride, sodium 3-(trimethylsilyl)-1-propanesulfonate, heptane, hydrochloric acid (aq, 37 %) and chloroform were purchased from Sigma-Aldrich (Taufkirchen, Germany). Borosilicate glass microscopy slides (Nexterion, Glass D) were purchased from Schott (Jena, Germany). The UV curing prepolymer NOA81 was purchased from Thorlabs (Dachau, Germany).

Figure 1: a) Micro flow reactors fabricated according to the described procedure (filled with blue ink for visualization), b) Microreactors are filled with luminescent probe in a photopolymer c) Small spots are polymerized using a 60x microscope objective and a 365 nm LED. d) Luminescence micrograph of temperature sensor spots inside the microchannel.

MICROREACTOR FABRICATION. Microreactors were prepared using photopolymerization of short-chain oligo ethylene glycol diacrylates between glass microscopy slides in a procedure related to earlier work.29,30 In short, fluidic access holes were powder blasted in borosilicate microscopy glass slides. The slides were pretreated with 3(trimethoxysilyl)propyl methacrylate in a heptane:chloroform (4:1) solution. A solution of 1 % (w/w) DMPA in OEG-DA was prepared and pipetted between the two glass slides. Adhesive tape was placed between the glass plates to obtain a defined channel height of 125 µm. A photomask with the fluidic structure was aligned on top of the assembly. After UV exposure, the unpolymerized oligomer was removed by aspiration through the access holes. Figure 1a shows a photograph of the fabricated microreactors that were employed throughout this work.


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Figure 2: Photothermal detection setup: (a-c) Modified inverted epifluorescence microscope (a) with additional upright light path (red) and second microscope objective (b) to focus the pump laser into the microchip. Epifluorescence light path (blue,c) is used to read out the luminescent temperature sensors in the channels. Schematic of the system components (d-j): The pump laser (d) is guided into the sample solution inside the microchannel. Downstream of the laser focus, a 40x microscope objective (e) collects luminescence from the integrated temperature sensor. Excitation light is provided by a high-power LED (f) modulated with a function generator (g). Emission light is collected by a photo multiplier tube (h) connected to a lock-in amplifier(i) A laptop (j) is used to control all electronic components.

TEMPERATURE SENSOR INTEGRATION. 400 mg of NOA 81 was diluted with 200 µL chloroform and doped with 0.8 mg of Ruthenium(II)-tris-(l,l0-phenanthroline) ditrimethylsilylpropane sulfonate (Ru(phe)3TMS2). Ru(phe)3TMS2 was prepared from the commercially available Ruthenium(II) tris(l,l0-phenanthroline) dichloride as described in Ref. 31. The sensor spots were integrated using a maskless photolithography technique.28 The microreactors were flushed with the prepolymer solution (Fig. 1b), positioned on an epifluorescence microscope (IX 51, Olympus, Hamburg, Germany) equipped with a high magnification objective (60x, NA 0.7, LCPlanFI, Olympus) and a near UV LED (365 nm, M365L2, Thorlabs). The field diaphragm and light path of the microscope were adjusted to yield even illumination in a small spot size. Short pulses of UV light (1 s) were then used to polymerize sensing spots inside the microreactor channels. The reactors were then rinsed with acetone and placed on a hotplate (100 °C, 1 h, Fig. 1c). The polymerization steps were carried out soon after filling the prepolymer mixture into the channels, because the chloroform in the coating solution deformed and dissolved the poly (ethylene glycol) channel walls after prolonged contact. Fig. 1d shows example temperature sensor spots. PHOTOTHERMAL DETECTION SETUP. For optical excitation, a frequency doubled Nd:YVO4 laser (Cougar, Time-Bandwidth Products, Zurich, Switzerland) with variable optical power up to 2 W (532 nm) was used. An epifluorescence microscope (iX71, Olympus) was equipped with a secondary upright light path above the specimen stage (Fig. 2a, from Thalheim Spezialoptik,

Thalheim, Germany). The pump laser was guided through the upper objective (20x, NA 0.4, LCPlanFl, Olympus) by means of a free space beam setup entering centered through the upright light path assembly (Fig. 2b). An electronic shutter was placed in the laser beam path enabling software control of laser exposure. The integrated luminescent temperature sensors were interrogated using the epifluorescence light path of the microscope (Fig. 2c). A 40x objective (NA 0.55, LCAch, Olympus) was focused on the sensor spots and a custom fluorescence filter cube was placed in the light path (Fig. 2e). The cube contained an excitation filter (BP420-480, Olympus), a dichroic mirror (LPD01-532RS-25, Semrock, Rochester, USA) and two emission filters. One emission filter was used to suppress the pump laser line (LP03532RS-25, Semrock), and the other to reduce the scattering background (BA590, Olympus). A high power LED MWCHL5 (Thorlabs, USA) was used for luminescence excitation (Fig. 2f) and a photomultiplier tube (PMT) H10723-20 (Hamamatsu Photonics, Herrsching, Germany) for luminescence detection (Fig. 2h). A lock-in amplifier (LIA) AFT-EL 205/2 (Anfatec Instruments, Oelsnitz, Germany) was used to read out the PMT. A function generator DZ1052 (RIGOL Technologies, China) was connected to the LED driver’s (DC2100, Thorlabs) external control connector to modulate the LED intensity with a 100 kHz sine wave (Fig. 2g). The synchronization output of the function generator and the PMT signal output were connected to the LIA using coaxial cables (Fig. 2i). In this way, the LIA can discriminate between the signal originating from the sensor luminescence and background noise. A laptop was employed for



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data acquisition and instrument control. Custom software was implemented in Python 2.7 to acquire data from the LIA, adjust the function generator’s output, and control the laser shutter (Fig. 2j). MISCELLANEOUS. Microreactors were connected to NemeSYS syringe pumps (cetoni, Korbussen, Germany) equipped with glass syringes (Innovative Labor Systeme, Stützerbach, Germany) by PEEK capillary tubing (id 150 µm, Vici Jour, Schenkon, Switzerland). The capillaries were sealed into the fluidic access holes using custom inhouse made PEEK clamps (see Fig. 2b) and suitable elastomeric ferrules and headless 6-32 PEEK screws (N-123-04 and N-123H, IDEX, Oak Harbor, USA). The absorption coefficient of the employed Amaranth dye was determined using a V650 spectrophotometer (Jasco, GroßUmstadt, Germany) to be 19941 +/- 96 L*mol-1*cm-1 at 532 nm.

RESULTS AND DISCUSSION TEMPERATURE SENSOR CALIBRATION. Micro flow reactors were fabricated with channel dimensions of 400 µm x 125 µm (WxH) and luminescent temperature spot sensors were integrated using the procedure outlined above. The integrated sensors were characterized with respect to their temperature response, size, and photostability. Using the maskless photopolymerization process, sensor spots as small as 26 ± 3 µm were obtained. The inset in Fig. 3 shows a luminescence micrograph of an example sensor spot array polymerized inside a microchannel, the dashed lines highlight the channel walls.

Figure 3: Temperature calibration of the luminescent tem2 perature sensors and linear fit (n=3, R = 0.99). Inset: Luminescent micrograph of temperature sensing spots in a microchannel (channel walls are illustrated by the dashed lines).

For calibration, the sensors were integrated into microchannels and the chip device mounted in a thermostat chamber enabling precise temperature control. The luminescence intensity of the sensor spots decreased with rising temperature. The data obtained were referenced to the values obtained at 18 °C (relative intensities). The relative luminescence intensity was found to be linear with temperature (Fig. 3, R2=0.99) with a slope of -0.90 ± 0.02 %*K-1. Considering the standard deviations of the signal over time the temperature resolution of the system was calculated as 0.3 K. The sensors showed moderate photobleaching with a decreasing signal intensity over

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time at -1.97 ± 0.05 %*min-1. Leaching of the temperature probe from the sensors spots was not observed within the experimental resolution. PHOTOTHERMAL SIGNAL. Using the integrated sensor spots the local photothermal heating of molecules in the microchannel was investigated. Suitable analytes for this kind of measurement need to absorb light at the wavelength of the pump laser. Here a green laser of 532 nm wavelength was employed. As a model analyte with a good absorbance, and negligible fluorescence quantum yield (Φ532 nm = 5.5*10-6),32 the azo dye Amaranth (E123) was employed. Aqueous solutions of dye were injected through an inlet and pumped through the microchannel structure at 20.0 µL*min-1 (6.66 mm*s-1) and at different concentrations. As shown in the graph Fig. 4a the luminescence sensors responded quickly to laser illumination producing a sharply decreasing intensity signal. When the laser illumination stopped, the luminescence intensity increased again (Fig. 4a). With no analyte present, at a concentration of 0 µM, a small background heating was observed, presumably caused by inelastic scattering or multiphoton absorption in water. This background signal corresponded to a local temperature increase of around 1.1 K (Fig. 4b). Since this source of background is constant at a particular constant laser power, it could be subtracted from the analyte-induced heating employing a suitable calibration curve.

Figure 4: Photothermal heating of a sample aqueous Ama-1 ranth solution (200 µM, 6.66 mm*s ,black curves) in a micro channel of 400 x 125 µm (W x H) upon laser illumination (532 nm, 2 W). Blue curves were acquired with pure water in the absence of analyte. a) Top: On and off cycles of the laser. Bottom: Luminescence intensity of the temperature sensor in the channel. green areas indicate laser illumination. b) Enlarged graph of luminescence intensity during a laser exposure. Markers denote the intensity values used for calculation of relative intensity. Black curves show the sensor response with 200 µM Amaranth in the channel. Blue curves show the sensor response during laser exposure in the absence of analyte (pure water).

To quantify the temperature increase, the relative intensity (R) of the sensor signal was determined by dividing the intensity after laser illumination (Ia) by the intensity (I0) before laser illumination (Fig. 4b). ூ ܴൌ ೌ Eq. 1


ூబ

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From this ratio, temperature differences (ΔT) were calculated using the data from the temperature calibration. Using relative intensity as the measurement signal is beneficial because it does not rely on absolute intensity values Absolute intensity is affected by drifts in ambient temperature and photo bleaching. Using the ratiometric signal described above, it was possible to overcome the photobleaching of the sensor spots and enable a more robust readout of temperature differences.

ple (Fig. 5a). A maximum was found at a linear velocity of 6.6 mm*s-1 (20 µL*min-1) and higher flow rates led to lower ΔT. This behavior can be explained with the offset of the heating zone and the downstream temperature sensors. Local heating in a channel will lead to symmetric temperature distribution around the heat source, which is altered by application of flow and the maximum temperature difference moves with the flow. The dependence of the photothermal signal on the applied laser power was investigated. The signal was found to increase linearly with increasing pump laser intensity within the range afforded by the pump laser (Fig. 5b). This behavior is expected for a linear optical absorption process. ΔT increased with increasing average optical power linearly (R2 = 0.99) up to 2 W with a slope of around 0.04 K*mW-1.

Figure 5: Measured temperature differences upon photothermal heating of aqueous Amaranth solution (n = 5). a) Influence of linear velocity of the analyte stream. b) Effect of laser power (average optical power) on the measured temperature difference in the microchannel.

SIGNAL CHARACTERIZATION. A variety of factors are expected to influence the photothermal signal from the sensor spots. Firstly, the optimal spatial location of the pump laser beam with respect to the temperature sensor spot was investigated. The sensor spot and the laser excitation had to be spatially offset in the channel to prevent damage to the sensor structures. Therefore, the pump laser focus was gradually moved away from sensor structures against the flow direction, until the generated background signal was low enough to enable sensors readout at maximum pump laser intensity. The optimum distance between sensor structures and pump laser focus in this setup was determined as approx. 300 µm. To investigate the flow rate dependence of the signal, the microreactor was flushed with Amaranth standards at a fixed laser power and a variable linear velocity from 0.6 mm*s-1 to 13 mm*s-1. Starting at stagnant solution the recorded ΔT rose with increasing linear velocity of the sam-

Figure 6: Calibration using standard solutions of amaranth. a) Decrease of sensor luminescence intensity in dependence of Amaranth concentration. Green box shows laser illumination b) Linear relationship between measured ΔT and Ama2 ranth concentration (n = 5, R = 0.99).

Using the optimized flow rate and the maximum laser intensity, the concentration dependence of the signal was investigated. A calibration was performed using aqueous Amaranth solutions. Fig. 6a compares the temperature responses generate by Amaranth solutions ranging from 0 µM to 400 µM. The results show a luminescence intensity decrease of the temperature sensor spot with increasing analyte concentrations. Calculating ΔT by evaluating the relative intensity for Amaranth solutions of different concentration, reveals a linear relationship between ΔT and amaranth concentration (Fig. 6b). The slope of the linear regression of the calibration was 171.7*10-3 ± 5*10-4 K*µM-1



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with a correlation coefficient of R2 = 0.99. The limit of detection (LOD) in this setup in terms of Amaranth concentration was determined from the weighted linear regression to be 13 µM at S/N = 3. This LOD corresponds to 3.2*10-3 AU and is comparable to values obtained with transmission-based techniques with enhanced path lengths using dedicated detection cells. For example, Ro et al.8 and Sieben et al.11 report 1.0*10-3 AU and 1.4*10-3 AU, using detection cells with 500 µm and 2.5 mm path lengths, respectively. Therefore, already in this initial study a sensitivity exceeding standard attenuated transmission-based absorbance detection was obtained. Some photothermal techniques were able to obtain lower detection limits, e.g. in thermal lens detection an LOD as low as 3.5*10-7 AU in aqueous media was reported.33 But considering that the results presented herein are the first of this concept, there is ample space for further development. The temperature resolution of the micro sensors herein was around 0.3 K whereas fluorescent or luminescent temperature sensors on larger scales have been reported to have a resolution as high as 0.01 K.34 In the future, advances in microfabrication or probe design could enable the use of integrated temperature probes with even better sensitivity for this application. The channel size and geometry could also be optimized in some formats because smaller channels can further increase the temperature differences induced in the solvent as simulated by Dennis et al.19 Similarly, reducing the gap between the integrated temperature sensors and the focus of the pump laser, that was thus far necessary to avoid damage to sensing structures, could be further reduced via choice of materials and temperature probes. REACTION MONITORING. After calibration and investigation of the absorbance sensitivity in a simple flowthrough system we investigated the suitability of the system for in-line detection of products in a microreactor. We chose the model reaction of sulfanilamide (4aminobenzenesulfonamide) and N-(1-Naphthyl)ethylene diamine (NED) in nitrous acid (NaNO2 in dilute HCl). This reaction is often used for photometric detection of nitrite. A microreactor with multiple inlets (Fig. 7a) was used to separate the two different steps of the reaction, namely diazotation and azo coupling. The reactor was supplied with sulfanilamide and nitrite for the diazotation in the first mixing structure. Subsequently NED was added to the reaction mixture in a second mixing structure for formation of the azo dye. Sulfanilamide and NED were supplied in excess (20 mM) and sulfanilamide was dissolved in dilute hydrochloric acid 5% (v/v) to facilitate diazo coupling. The total volumetric flow rate was 20 µL*min-1 with sulfanilamide and the dissolved sodium nitrite at 5 µL*min-1 each, and the NED solution at 10 µL*min-1. Fig. 7b shows the results of probing the reaction mixture close to the outlet of the microreactor. The measured temperature difference was increasing with the concentration of nitrite injected into the reactor.

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Figure 7: Monitoring the formation of an azo-dye in a micro flow reactor by photothermal absorbance detection. a) The starting materials are fed to the reactor in two steps. Sulfanilamide in acidic solution and NaNO2 are added first to perform diazotation. Coupling partner NED is added in the second mixing structure. The effluent of the second mixing structure is probed by laser illumination and temperature measurement. b) Measured ΔT increased with increasing NO2 concentration. The data were fitted with a hyperbola 2 (R =0.98).

These flow rates led to a short total residence time in the reactor of approximately 75 s. Therefore, the detected ΔT was not linear, presumably because the reaction was not quantitative. The data, however nicely confirm the capability of the detection method for in-line monitoring in real time. The data were best fitted with a hyperbola (ΔT = 99°C*cNO2-/(1010 µM + cNO2-), P1 = 99 °C, P2= 1010 µM, R2 = 0.98). The resulting graph shows a limit of detection for nitrite of 26 µM (S/N = 3) with a dynamic range extending to at least 1 mM at a total assay time of only 75 s. Since the focus here was on real-time reaction monitoring, lower detection limits appear possible with longer reaction times.

CONCLUSION Luminescent micro temperature sensors were integrated into microfluidic channels using maskless photopolymerization. These integrated sensors enabled quantification of optical absorbance at 532 nm based on photothermal heating of analytes upon laser excitation. In contrast to other detection setups based on the photothermal effect, our system does not rely on the refractive index or viscosity changes but directly probes the local solvent temperature. We characterized the detection system and


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showed that linear calibration curves were obtained for aqueous solutions of the food dye Amaranth, which was employed as model compound. The detection limit in the present setup was found to be 3.2*10-3 AU, which is comparable to on-chip absorbance detection based on attenuated light transmission with optical path length that are at least one order of magnitude longer. The detection scheme was successfully used in monitoring of an azo-dye synthesis in a microreactor and was able to detect nitrite in the lower micromolar range in less than two minutes. As a drawback, the technique is dependent on moderately high power laser light sources with the appropriate wavelengths. The method could also prove useful for absorbance quantification in highly scattering media. Other promising fields of application are nanofluidic systems or spatially resolved absorbance detection.

AUTHOR INFORMATION Corresponding Author * Stefan Nagl, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China, Tel. +852-3469-2629, e-mail: [email protected], http://naglgroup.ust.hk Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS The authors were partially supported by the German Research Foundation (DFG, NA947/1-2 and 2-1) which is gratefully acknowledged.

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Figure 1: a) Micro flow reactors fabricated according to the described procedure (filled with blue ink for visualization), b) Microreactors are filled with luminescent probe in a photopolymer c) Small spots are polymerized using a 60x microscope objective and a 365 nm LED. d) Luminescence micrograph of temperature sensor spots inside the microchannel. 190x208mm (96 x 96 DPI)


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Figure 2: Photothermal detection setup: (a-c) Modified inverted epifluorescence microscope (a) with additional upright light path (red) and second microscope objective (b) to focus the pump laser into the microchip. Epifluorescence light path (blue,c) is used to read out the luminescent temperature sensors in the channels. Schematic of the system components (d-j): The pump laser (d) is guided into the sample solution inside the microchannel. Downstream of the laser focus, a 40x microscope objective (e) collects luminescence from the integrated temperature sensor. Excitation light is provided by a high-power LED (f) modulated with a function generator (g). Emission light is collected by a photo multiplier tube (h) connected to a lock-in amplifier(i) A laptop (j) is used to control all electronic components. 354x215mm (96 x 96 DPI)



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Figure 3: Temperature calibration of the luminescent temperature sensors and linear fit (n=3, R2 = 0.99). Inset: Luminescent micrograph of temperature sensing spots in a microchannel (channel walls are illustrated by the dashed lines). 185x127mm (96 x 96 DPI)


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Figure 4: Photothermal heating of a sample aqueous Amaranth solution (200 µM, 6.66 mm*s-1, black curves) in a micro channel of 400 x 125 µm (W x H) upon laser illumination (532 nm, 2 W). Blue curves were acquired with pure water in the absence of analyte. a) Top: On and off cycles of the laser. Bottom: Luminescence intensity of the temperature sensor in the channel. green areas indicate laser illumination. b) Enlarged graph of luminescence intensity during a laser exposure. Markers denote the intensity values used for calculation of relative intensity. Black curves show the sensor response with 200 µM Amaranth in the channel. Blue curves show the sensor response during laser exposure in the absence of analyte (pure water). 518x302mm (96 x 96 DPI)



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Figure 5: Measured temperature differences upon photothermal heating of aqueous Amaranth solution (n = 5). a) Influence of linear velocity of the analyte stream. b) Effect of laser power (average optical power) on the measured temperature difference in the microchannel. 190x258mm (96 x 96 DPI)


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Figure 6: Calibration using standard solutions of amaranth. a) Decrease of sensor luminescence intensity in dependence of Amaranth concentration. Green box shows laser illumination b) Linear relationship between measured ∆T and Amaranth concentration (n = 5, R2 = 0.99). 158x236mm (96 x 96 DPI)



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Figure 7: Monitoring the formation of an azo-dye in a micro flow reactor by photothermal absorbance detection. a) The starting materials are fed to the reactor in two steps. Sulfanilamide in acidic solution and NaNO2 are added first to perform diazotation. Coupling partner NED is added in the second mixing structure. The effluent of the second mixing structure is probed by laser illumination and temperature measurement. b) Measured ∆T increased with increasing NO2- concentration. The data were fitted with a hyperbola (R2=0.98). 171x258mm (96 x 96 DPI)


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236x119mm (96 x 96 DPI)


On-Chip Photothermal Analyte Detection Using Integrated

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