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Cite this: DOI: 10.1039/x0xx00000x

Fusion of Microlitre Water-in-Oil Droplets for Simple, Fast and Green Chemical Assays S.-H. Chiua and P. L. Urbana,b

Received 00th January 2012, Accepted 00th January 2012 DOI: 10.1039/x0xx00000x www.rsc.org/

A simple format for microscale chemical assays is proposed. It does not require the use of test tubes, microchips or microtiter plates. Microlitre-range (ca. 0.7 – 5.0 µL) aqueous droplets are generated by commercial micropipette in a non-polar matrix inside a Petri dish. When two droplets are pipetted nearby, they spontaneously coalesce within seconds, priming a chemical reaction. Detection of the reaction product is accomplished by colorimetry, spectrophotometry, or fluorimetry using simple light-emitting diode (LED) arrays as the sources of monochromatic light, while chemiluminescence detection of the analytes present in single droplets is conducted in the dark. Smartphone camera is used as detector. The limits of detection obtained for the developed in-droplet assays are estimated to be: 1.4 nmol (potassium permanganate by colorimetry), 1.4 pmol (fluorescein by fluorimetry), 580 fmol (sodium hypochlorite by chemiluminescence detection). The format has successfully been used to monitor the progress of chemical and biochemical reactions in time with sub-second resolution. Semi-quantitative analysis of ascorbic acid using Tillman’s reagent is presented. A few tens of individual droplets can be scanned in parallel. Rapid switching of the LED light sources with different wavelengths enables spectral analysis of multiple droplets. Very little solid waste is produced. The assay matrix is readily recycled, thus the volume of liquid waste produced every time is also very small (typically, 1 – 10 µL per analysis). Various water-immiscible translucent liquids can be used as the reaction matrix: including silicone oil, 1-octanol as well as soybean cooking oil.

1. Introduction Traditionally, chemical reactions are conducted on large volumes of solvents, which inevitably leads to production of large amounts of chemical waste. In the 21st century, environmental protection has become a major goal. In the field of analytical chemistry efforts are constantly made to develop assays characterized by little consumption of chemicals and low environmental impact. This trend can be referred to as “green analytical chemistry”.1,2 Miniaturization of analytical methods can also decrease the costs, and increase availability of chemical assays in the resource-limited regions.3 There exist various ways to conduct chemical reactions in the microscale, for example using: flow injection analysis,4 microfluidic systems,5-7 microcentrifuge tubes,8 microtiter plates,9 and even plastic containers made of packaging material.3 The implementation of instrumental techniques is costly while using disposable plastic vessels inevitably leads to production of substantial amounts of chemically contaminated solid waste. Moreover, handling microlitre and sub-microlitre volumes of samples in the open containers does not prevent solvent evaporation, which can lead to systematic and random errors. Emulsions are mixtures of immiscible liquids with different polarities. They exemplify compartmentalized chemical systems.10 While they are easy to prepare, emulsions find applications in various fields of science and industry, including

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food chemistry,11,12 medicine,13-16 firefighting,17 metallurgy,18 cosmetic production19 and even single-molecule measurements.20 For instance, a suspension of microscopic aqueous droplets in oil can be a matrix for chemical reactions such as synthesis of imine compounds21 or Diels-Alder reaction.22 The volumes of individual emulsion droplets are very small, often in the picolitre range.23-25 Therefore, they are too small to be addressed individually using conventional liquid handling tools such as pipettes. On the other hand, nanolitre or even picolitre volume droplets – dispersed in immiscible liquid matrices – can be processed in microfluidic systems,26-28 and they can be treated as individual microscale “test tubes”. However, microfluidics has not yet become a universal platform for chemical and biochemical analysis because it often requires a high level of expertise and specialized instrumentation. There is no convenient format which would allow one to carry out reactions and analytical measurements in the volumes smaller than those accommodated by microcentrifuge tubes or microtiter plates routinely, using conventional laboratory toolkit. Here we propose a simple, inexpensive, and environmentfriendly method of conducting chemical assays in microlitre and sub-microlitre volumes. It relies on utilization of aqueous droplets suspended in a non-polar immiscible matrix as assay vessels with adaptable volume. These droplets can be produced by micropipette or syringe. Two or more droplets can readily be

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Analyst ARTICLE

Journal Name DOI: 10.1039/C5AN00847F

merged to initiate a chemical reaction without an active supply of energy. This method is compatible with various kinds of optical detection (colorimetric, fluorimetric, and luminescence) enabled by inexpensive equipment (smartphone camera, lightemitting diode (LED) light source). The assay matrix (e.g. silicone oil) can be recycled and reused. The mixing of the solutions present in the substrate droplets and subsequent detection is very fast, and the assays can readily be multiplexed.

2. Experimental section 2.1. Materials Ammonium acetate, brilliant blue G, copper sulphate pentahydrate, D-glucose, 2,6-dichloroindophenol sodium salt hydrate, ferroin, fluorescein, fluorescein isothiocyanate-casein (FITC-casein), L-ascorbic acid, luminol, myoglobin (from equine skeletal muscle), methylene blue, 1-octanol, and trypsin (from bovine pancreas) were all purchased from Sigma-Aldrich (St Louis, MO, USA). Silicone oil (AR 20) was purchased from Uni-Onward (Taipei, Taiwan). Sodium hydroxide and LCgrade water were purchased from Merck (Darmstadt, Germany). Potassium permanganate was purchased from Avantor (Center Valley, Philadelphia, Pennsylvania, USA). Household bleach (6% sodium hypochlorite) was purchased from A-Mart (Far Eastern, Taipei, Taiwan). Soybean oil was from Uni-President (Tainan, Taiwan). Blue and red ink were from Simbalion (Taipei, Taiwan).

Figure 1. Chemical assays in microlitre-scale water-in-oil droplets. (A) Microlitre and sub-microlitre droplets of aqueous solutions are dispensed by micropipette into a Petri dish filled with oil matrix. (B) Experimental setups for readout of microdroplet contents by (i) colorimetry (cf. Figure 2A); (ii) fluorimetry (cf. Figure 2B); and (iii) chemiluminescence detection (cf. Figure 2C). (C) Schematic representation of droplet merger in silicone oil matrix (cf. Figure 3).

2.2. Equipment The in-droplet reactions were performed using few pieces of common equipment (Figure 1A). The assays were conducted inside a glass Petri dish (∅ = 3.6 cm). The container was modified by coating the inner walls with Cytop CTL-809M (AGC; Asahi Glass, Ibaraki, Japan) diluted with Solv-180 (AGC) diluent in the ratio 1:10 (v/v). This way, the inner surface of the Petri became hydrophobic, which prevented splashing of the aqueous droplets. A 1-mL aliquot of the Cytop solution was deposited on the bottom of a clean Petri dish, and heated on hot plate under fume hood at 180 °C for 1 h. The Petri dish was cooled down, and filled with 3 mL of silicone oil. It was placed on the surface of the observation stage (Figure 1B).

1B-(i)). The intensity of light above the surface of the tracing paper was ∼ 4000 lux (Table S1). The spectrum of the white light source was recorded using a fibre optic spectrophotometer (USB4000-VIS-NIR; Ocean Optics, Dunedin, FL, USA; Figure S1H). In the case of fluorimetric detection, the Petri dish was placed on black paper. The light source consisted of 19 blue LEDs (type, nominal voltage 12 V, operated at 9.0 V; Centenary, Hsinchu, Taiwan), and it was positioned above the sample (distance: ~ 7 cm) near the camera. Its spectrum is shown in Figure S1I. The angle between the support surface and the axis of the light source was 30° (Figure 1B-(ii)). In the case of chemiluminescence detection, the Petri dish was placed on black paper. This time, no light source was used (Figure 1B-(iii)). Other light sources were used to perform the measurements of the multi-wavelength, the homemade device 2.3. Generation of droplets and initiation of reaction Aqueous droplets in oil were produced by injecting 0.7 or 5 µL contains 14 LEDs; 2 for each of 7 wavelengths (cf. Table S1 of various aqueous solutions into the oil matrix inside Petri dish. and Figure S1). RGB values obtained by digital imaging systems can be The droplet precipitated onto the bottom of the Petri dish within 29-33 Smartphones are < 10 s. However, due to the hydrophobic nature of the Cytop- utilized to carry out chemical analyses. 34-42 In fact, the coated glass surface, it did not splash; it preserved its spherical increasingly used in portable analytical devices. camera of smartphone can be used as an optical sensor in shape. The droplets could also be generated by inserting one chemical assays. Also in this study, most data were acquired end of fused silica capillary (ID 150 µm, OD 375 µm; 101032132, GLScience, Tokyo, Japan). When a droplet was using a commercial smartphone (iPhone 5; Apple, Cupertino, produced in the close proximity of another droplet, the two CA, USA) and its built-in camera (8 megapixels, 1080P Full droplets automatically merged and mixed within few seconds. HD). Raw images were saved in JPG files, while videos were All experiments were conducted at ambient temperature: ∼ 21 saved in MOV files. In the case of the MOV files, selected frames were subsequently exported to JPG files. The JPG files °C. were transferred to a computer and analysed using the ImageJ (ver. 1.48; National Institutes of Health, Bethesda, MD, USA). 2.4. Detection and data treatment In the case of colorimetric (light absorption) detection, the In the case of colorimetric measurements, for every droplet, observation stage was made of a transparent acrylic box (l/w/h RGB values of few tens of pixels (~ 20) were obtained. 160/120/30 mm). It was covered with one layer of tracing paper Considering that the droplets have spherical shape, the values (thickness: 100 µm). A white LED array light source (6 × 8 of these pixels correspond to apparent maximum optical LEDs, type, nominal voltage 12 V, operated at 9 V; Yi Yang, pathlengths of ∼ 0.9, 1.2, or 3 mm, in the case of the nominal volumes 0.7, 1.4, and 10 µL, respectively. The values of the Hsinchu, Taiwan) was placed on the surface of the box (Figure selected RGB channels (obtained for every droplet) were used

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Journal Name as intensities (I) of light in the following calculations. Absorbances were computed using the formula:43,44 A = log ቀ

ூబ /ூ

ூబష౨౛౜ /ூ౨౛౜

ቁ,

eq. 1

where I is the intensity of signal obtained for a droplet, I0 is the intensity of signal obtained for adjacent pixels, Iref is the intensity of signal obtained for a solvent droplet (blank), and I0ref is the intensity of signal obtained for the pixels adjacent to the solvent droplet. Considering the spectral characteristics of the transmitted or emitted light, in the colorimetric and fluorimetric measurements, we only extracted the values of the G channel. Additionally, in fluorimetric measurements, the background value was subtracted. In the chemiluminescence measurements, we used the values of the B channel without further pre-treatment of the collected data.

3. Results and discussion 3.1. Characterization of the in-droplet assay In order to characterize the detection system for the analysis of individual water-in-oil droplets, we carried out a number of experiments and measurements. The chosen droplet volume was a compromise between different factors: it should be as small as possible to achieve low consumption of reagents and – at the same time – enable confinement of a few tens of droplets in a few square centimetre area of Petri dish but it should be large enough to enable precise pipetting and sensitive optical detection. The repeatability of pipetting individual droplets was determined when pipetting 20 individual droplets and measuring diameters of the corresponding spots in the digital image (Figure S2). The average diameter of 0.7 and 1.4 µL droplet footprints generated by pipette was 38.33 ± 1.11 and 49.93 ± 1.57 pixels (n = 20). The accuracy of droplet volume was determined by weighing 20 droplets on an analytical balance. The calculated volumes were 0.57 ± 0.02, 1.24 ± 0.04 and 5.15 ± 0.15 µL for the nominal volumes 0.7, 1.4 and 5.0 µL respectively. This result indicates the systematic errors of -18, 11 and +3% in the case of pipetting such low volumes of liquid. Although pipetting 0.7 and 1.4 µL droplets resulted in high absolute errors, for the sake of simplicity, here we report nominal volumes of droplets. However, the mass limits of detection are calculated based on the accurate volumes. Both experiments (measuring diameters of spots in images and weighing single droplets) show good precision of the droplets generated by pipette in the oil matrix (relative standard deviations: 2.9 or 3.5% for 0.7 µL droplets; 3.1 or 3.2% for 1.4 µL droplets). Subsequently, we have verified the possibility to implement colorimetric, fluorimetric and chemiluminescence detection to quantify the contents of droplets using smartphone camera. In the case of colorimetric detection, solutions of potassium permanganate in water were used as standards. Three 1.4-µL droplets were generated for every concentration (×6) in the range (0-100 mM). The obtained absorbance values were fitted with the equation: A = (0.108 ± 0.007) C – (0.138 ± 0.043) .

eq. 2

The resulting calibration plot (Figure S3A) shows satisfactory linearity of the assay (coefficient of determination: R2 > 0.98). The LOD of this colorimetric measurement was estimated to be

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1.19 mM (~ 1.4 nmol at 1.1 µL). In the case of fluorimetric detection, fluorescein was used as standard. The calibration equation (Figure S3B) was obtained for the concentration range 0 – 15 µM: I = (12.3±0.8) C + (57.8±5.6).

eq. 3

The coefficient of determination (R2) was > 0.97. The LOD of the fluorimetric measurement was estimated to be 1.37 µM (∼ 1.4 pmol at 10.3 µL). In the case of chemiluminescence detection, droplets containing 0 – 20 mM sodium hypochlorite (analyte) were reacted with luminol (reagent) droplet. The data points were fitted with the following non-linear function (Figure S3C): I = 5.8 +

஺஼

஼ା஻

,

eq. 4

where the I is the intensity obtained from the chemiluminescence signal; A and B are the fitting parameters. The LOD of the chemiluminescence measurement was estimated to be 112 nM (∼ 580 fmol at 5.15 µL). In analytical chemistry, there are cases where either concentration or mass LODs are important – depending on the expected concentration of analyte in the sample and the sample volume available for analysis. Here, the concentration LODs can be decreased by increasing droplet volume (and increasing the optical pathlength), while the mass LODs can be decreased – to some extent – by decreasing droplet volume. Therefore, the proposed format can readily be tuned according to the specific analytic requirements. While smartphone camera is an accessible detector for chemical assays, the image pre-treatment conducted by the built-in processor may compromise quality of the analytical data.36 Nevertheless, it is pleasing to note that the calibration plots obtained here enable quantitative analysis using three different detection modes (Figure S3) – at high and low light levels. 3.2. Monitoring in-droplet reactions Two droplets (0.7 – 5.0 µL) dispensed into the oil matrix in the close proximity spontaneously merge within a few seconds (Figures 1C and 3A). This is due to disorganisation of molecular arrangement of the matrix in the contact zone between two droplets, formation of a aqueous-phase junction, and action of hydrostatic and hydrodynamic forces that push the contents of the droplets toward each another. The resulting turbulence, convection, diffusion, and the development of reaction fronts may further contribute to mixing of the reactants. The ability to merge the droplets containing different solutions in a short period of time allowed us to prime in-droplet reactions, and to monitor reaction progress over time. In the first example, we implemented colorimetric detection to follow reduction of manganese from permanganate substrate according to the two-step process:45,46 MnO4– + e– → MnO42– MnO42– + 2H2O + 2e– → MnO2 + 4OH–

eq. 5 eq. 6

Figure 2A shows the change in absorbance (G channel) over time. This change mainly represents reduction of permanganate 7+ to 6+ (eq. 5). Here, glucose is used as an ancillary reagent due to its aldehyde groups which are oxidized by permanganate in the alkaline environment. Manganese species with different oxidation states absorb light at different wavelengths;47,48 thus, it is possible to distinguish them based

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Figure 3. Merging microlitre-scale aqueous droplets suspended in oil matrix. (A) Two 0.7-µL droplets containing potassium permanganate and the mixture of glucose and sodium hydroxide are merged (cf. Figure 2A). (B) (i) Initially, a 0.7-µL droplet of potassium permanganate solution is merged with a 0.7-µL droplet of glucose solution. (ii) Subsequently, a 0.7-µL droplet of sodium hydroxide is added, which initiates the oxidation of permanganate. Scale bars: 1 mm.

on the transmitted light images obtained using the smartphone camera. In this case, selectivity is due to the spectral characteristics of the microfilters in the camera’s complementary metal oxide semiconductor (CMOS) chip. In the second example, fluorescein isothiocyanate (FITC)casein conjugate was reacted with trypsin. In FITC-casein, fluorescence is quenched by amino acid residues.49,50 The hydrolysis of FITC-casein catalysed by trypsin led to the increase of fluorescence intensity (Figure 2B). Finally, the reaction of luminol oxidation51,52 was monitored by chemiluminescence (Figure 2C). In that case, the sampling rate was 5 data points per second (greater than in the case of colorimetric and fluorimetric assays) to adapt to the short duration of photon burst. In fact, the temporal resolution of detection depends on the type of the camera chip used; using the smartphone camera, approximately 30 values can be extracted every second, if required. Moreover, using the water-in-oil droplets, not only the single-step (Figure 3A) but also multi-step chemical processes could be performed. To demonstrate this feature, we mixed three droplets containing the components of the permanganate reaction sequentially (Figure 3B). Initially, permanganate droplet was merged with glucose droplet. After adding the third droplet containing sodium hydroxide, reduction of manganese could be observed. Figure 2. Application of the proposed method in the monitoring of chemical reactions in microlitre volumes by (A) colorimetry (oxidation of permanganate), (B) fluorimetry (hydrolysis of FITCcasein), and (C) chemiluminescence detection (oxidation of luminol). In (A), white LED source was used (λmax = 440 and 547 nm; cf. Figure S1H). In (B) blue LED light source was used (λmax = 475 nm; cf. Figure S1I). In (C), the monitoring was conducted in the absence of light. In (A), the final reaction volume was ∼ 1.4 µL, while in (B) and (C), the final reaction volumes were ∼ 10 µL. Reagents in (A): 10 mM potassium permanganate; 222 mM glucose mixed with 167 mM sodium hydroxide. Every data point in (A) is obtained from average of ~ 30 pixels while in (B) and (C) ~ 65 pixels. Data point symbols in (B): , a droplet containing FITCcasein merged with a droplet containing 20 mM ammonium acetate (control); , a droplet containing FITC-casein merged with a droplet containing 59.4 mg mL-1 trypsin dissolved in 20 mM ammonium acetate (reaction). Data point symbols in (C) represent different concentrations of NaOCl: , 0 mM (control); , 1 mM; , 4 mM; , 8 mM; , 20 mM; , 60 mM; , 100 mM. The video records of these experiments are included in the E.S.I.: (A) Movie S1 – colorimetric monitoring of permanganate reaction; (B) Movie S2 – fluorimetric monitoring of FITC-casein hydrolysis by trypsin; (C) Movie S3 – chemiluminescence monitoring of the reaction of 100 mM NaOCl with luminol.

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3.3. Assay matrix Apart from silicone oil, we also investigated the suitability of other immiscible media, including soybean oil and 1-octanol, for the in-droplet assays. All these matrices are suitable for priming reactions by fusion of aqueous droplets. Figure S4 shows reaction progress curves obtained for the three tested matrices. Since the mixing of the droplet contents relies on the surface tension and hydrostatic pressure exerted during the merger, the initial progress of the reaction is slightly affected by the change of the assay matrix. Replacement of silicone oil with vegetable oil makes this system suitable for chemical analysis in resource-limited areas. The yellow colour of vegetable oil may slightly affect the performance of analyses involving species absorbing or emitting light at some wavelengths. Please note that the oil matrix can readily be separated from the aqueous phase droplets, and reused. Thus, only small volume of chemical waste is produced. Here, we used a metal spatula or small pieces of cellulose tissue to remove water droplets from the oil matrix. However, one can also implement special filiters53 to separate oil and the aqueous waste left over many in-droplet assays.

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ARTICLE DOI: 10.1039/C5AN00847F

3.4. Spectral scan of multiple water-in-oil droplets LEDs are often used in analytical chemistry as the source of monochromatic light.54 In fact, diodes emitting light at different wavelengths55,56 and liquid crystal displays with LED back illumination57 have proved to be useful in spectral analysis of liquid samples in microscale. Thus, it is appealing to use this detection scheme for simultaneous spectral analysis of multiple droplets suspended in the immiscible matrix. To achieve this goal, we assembled a simple source of light which consists of 7 types of diodes emitting light at different wavelengths (Table S1, Figure S1). The LEDs were powered from a direct current power supply via resistors selected separately for each type of LED. They were triggered by a relay board operated using the Denkovi Relay Manager software (ver. 1.4; Denkovi Assembly Electronics, Byala, Bulgaria). The iPhone camera as well as the Digital Single Lens Reflex (DLSR) camera (EM-1, Olympus, Tokyo, Japan) were used as detectors in this experiment. In each case, the camera was set 8 cm above the polycarbonate box fitted with the LED source. The wavelength presentation time was set to 4 – 6 s for the iPhone, and 0.5 s for the DLSR camera. This time setting is due to the speed of intensity balance conducted by the microprocessor of the device. Snapshots of multiple droplets suspended in silicone oil were taken as the wavelengths changed and the image intensity stabilized. This detection procedure resulted in a number of images revealing the light absorption properties of the droplet contents (Figure S5). Droplets appear dark on the images taken at light wavelengths corresponding to the absorption maxima of the encapsulated analytes. For example, potassium permanganate and ferroin absorb light at 397, 465, 525, and 586 nm, while myoglobin absorbs light at 397 nm (Figure S5A). In this mode of detection, selectivity is not only due to the microfilters (red, green, and blue) of the camera chip but mainly due to the wavelength of LED light transmitted through the Petri dish. This proof-of-concept experiment suggests that it may be possible to carry out spectral analysis of multiple droplets at the same time in just a few seconds, and gives the promise for multiplexing various chemical assays using the water-in-oil droplet format. 3.5. Detection of ascorbic acid in beverage To further verify the usefulness of this method in the analysis of complex samples, we applied it in semi-quantitative analysis of ascorbic acid in tea infusion matrix. Ascorbic acid reduces Tillman’s reagent following the equation:58

. eq. 7 This reaction leads to decolouration of blue assay mixture. Therefore, it is amenable to colorimetric detection. In the course of analysis, 5-µL sample droplets were fused with 5-µL Tillman’s reagent (1 mM 2,6-dichloroindophenol sodium salt hydrate in aqueous 200 mM ammonium acetate) droplets. In order to perform the semi-quantitative analysis, we prepared analyzed standard solutions from 0.1 mM to 1 mM. The infusion of oolong tea was prepared by incubating 1.42 g of tea leaves (Tung Ting; King Ping, Taipei, Taiwan) in 15 mL of water at ∼ 100 °C for 5 minutes. After cooling, the sample was spiked with ascorbic acid to the final concentration 5 mM. It was diluted 10× with pure water prior to analysis. The assay droplets were illuminated by 633-nm home-made red LED source (Figure S6). Pixel intensities (G) were measured 15 s after the droplets merged, and processed according to the eq. 1.

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We treated the raw data using two different methods. In the first method, we measured pixel intensities along a diagonal line within the droplet footprint (I), and a similar line outside the droplet (Iref). Following calibration, the concentration of ascorbic acid in the original tea sample was estimated to be 5.2±2.8 mM. In the second method, we averaged intensities of pixels along 10 diagonal lines within the droplet footprint (I) and 10 lines of comparable length around the droplet footprint (Iref). This time, the concentration was estimated to be 7.9±1.2 mM. Thus, this method may be considered qualitative or semiquantitative. For example, it may facilitate rapid screening of beverage samples fortified with ascorbic acid. Please note that the systematic and random errors may be associated with the non-uniform illumination of the assay container due to the implementation of the home-made LED source as well as measurement instabilities resulting from the deployment of the automated balance function41 in the smartphone. In future, one can further improve this system by changing the light source and modifying the smartphone to eliminate the auto-balance feature.

4. Conclusions This study demonstrates the possibility to perform various chemical analyses using droplets obtained by pipetting aqueous solutions into oil matrices. The format is compatible with colorimetric, fluorimetric, and chemiluminescence detection. Spectral analysis of droplets is possible. It can be combined with various reaction-based assays, in which case the analytical selectivity is due to the chosen reaction system, and comparable with classical formats (test tube, microtiter plate). It is amenable to multiplexing, and it can provide temporal information on chemical processes taking place in the droplets. It limits consumption of samples and precious assay reagents. It would be appealing to further combine this format with other detection schemes, including turbidimetry, infrared and Raman spectroscopy. The proposed approach generates very small volumes of chemical waste, and produces modest amounts of solid waste (pipette tips). It uses very few pieces of inexpensive hardware (Petri dish, acrylic box, LED source, smartphone, and a low-end computer). It does not require microfabrication. Thus, the proposed method follows the trend of green analytical chemistry, and it can readily be implemented in resourcelimited regions. The method is applicable to reactions in aqueous phase. The immiscible matrix needs to be compatible with the reaction and detection; different oils can be used. The oil separates the reaction microenvironment from the atmospheric air, which limits evaporation of sample solvent and/or oxidation of unstable reactants due to contact with oxygen. The assay components do not have contact with solid materials (e.g. polystyrene, glass, polydimethylsiloxane), which can facilitate handling biomolecules – some of which tend to adsorb on surfaces. However, it must be assured that the analytes and reagents do not partition to the oil phase. Moreover, the small droplet size and the large surface-tovolume ratio make heat dissipation faster and better defined than it is in the case of larger and open reaction vessels (e.g. wells of microtiter plates). This feature can enhance thermal control of exothermic and endothermic processes taking place in the droplets.

Acknowledgements Thanks are due to Prof. Yu-Chie Chen for lending us the Ocean Optics spectrometer. We also thank the Ministry of Science and

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Technology of Taiwan (formerly, National Science Council; Grant no. NSC 102-2113-M-009-004-MY2) for the financial support of this work.

Notes and references a

Department of Applied Chemistry, National Chiao Tung University 1001 University Rd, Hsinchu, 300, Taiwan b Institute of Molecular Science, National Chiao Tung University 1001 University Rd, Hsinchu, 300, Taiwan Electronic Supplementary Information (ESI) available: text file (including Table S1 and Figures S1-S6), and three video files (Movies S1-S3). See DOI: 10.1039/b000000x/ 1 2 3 4 5 6 7 8

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Fusion of microlitre water-in-oil droplets for simple, fast and green chemical assays.

A simple format for microscale chemical assays is proposed. It does not require the use of test tubes, microchips or microtiter plates. Microlitre-ran...
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