The authors would like to convey their gratitude for the technical support provided by National Synchrotron Radiation Research Center (NSRRC) (Taiwan). The authors are grateful for the financial support provided by the Ministry of Science and Technology of the Republic of China (Taiwan) and the Industrial Technology Research Institute (Taiwan).

Caution! Several chemicals (e.g., acrylamide, 1,1&#39;-dichloroethene) used in these procedures are acutely toxic and carcinogenic. Consult all relevant material safety data sheets (MSDS) prior to use. Follow appropriate safety practices when performing the experiments.
Note: Unless otherwise stated, carry out all procedures at ambient temperature in a class 100 laminar flow hood.
1. Fabrication of the Dipole-assisted SPE Microchip
<ol>
	<li>Preparation of the PMMA Microchip 
	Note: The fabrication protocol of the chip was similar to that described elsewhere.8
	<ol>
		<li>Draw the network pattern of the chip (Figure 1(a)) using computer aided design (CAD) software according to the manufacturer&#39;s protocol.</li>
		<li>Mount a PMMA sheet (350 mm (L) x 20 mm (W) x 2 mm (H)) on the working table of the laser micromachining system and then focus the laser source on the surface of the PMMA sheet.</li>
		<li>Select the Print in the CAD software and then set the Power, Speed, and Pen Mode as 45% (4.5 W), 13% (99.06 mm sec&#8722;1), and VECT through the control panel of the micromachining system. 
		Note: Parameters such as Power, Speed and Pen Mode affecting the channel features were investigated in advance. The evaluation method was similar to that proposed by Yuan and Dasa.17 The parameters selected in this study were used to machine an appropriate channel for adaption to conduits without complicated academic purposes. People can select another condition for laser machining in accordance with their need.</li>
		<li>Print the drawn pattern and then machine the PMMA sheet by the laser micromachining system according to the manufacturer&#39;s protocol. Figure 1(b) displays the layout of the fabricated microchip. Figure 1(c) displays the photograph of the cross-section of the machined plate. 
		Caution! Wear goggles when using the laser system to avoid severe eye damage due to exposure to the laser radiation. An appropriate exhaust system is recommended because of the production of fumes/smoke during laser machining.</li>
		<li>Drill three 1/16-inch diameter access holes for a sample inlet, a buffer inlet, and an eluent inlet on the bottom plate and one for a confluent outlet on the cover plate Figure 1(b). 
		Caution! Avoid body contact with the drill bit during the machining procedures to prevent physical injury. Wearing gloves is prohibited when drilling.</li>
		<li>Immerse the machined plates into 1 L of 0.1% (w/v) sodium dodecyl sulfate (SDS) solution in 1-L beaker with agitation via an ultrasonic oscillator for 10 min.</li>
		<li>Replace the SDS solution with deionized water and agitate via an ultrasonic oscillator for 10 min.</li>
		<li>Replace the residual D. I. H2O with fresh one and then immerse the machined plates in 1 L of D. I. H2O with agitation via an ultrasonic oscillator for 10 min. Afterward, dry each cleaned plate with a gentle stream of nitrogen for 2 min.</li>
		<li>Align the two machined plates with the naked eye and then sandwich the two plates between two glass boards by using binder clips.</li>
		<li>Bond the two plates under compression at 105 &#176;C for 30 min.</li>
		<li>Cool the sandwich to ambient temperature and then remove the binder clips and glass boards.</li>
		<li>Insert 1/16-inch outer diameter poly(etheretherketone) (PEEK) tubes into the access holes and then secure the conduits with a two-component epoxy-based adhesive.</li>
		<li>Dry the adhesive at ambient temperature for 12 hr.</li>
	</ol>
	</li>
	<li>Modification of the Channel Interior of the PMMA Microchip 
	Note: The following partially refers to published procedures with slight modifications.8,18,19
	<ol>
		<li>Deliver a saturated sodium hydroxide (NaOH) solution at a flow rate of 100 &#181;l min-1 via a peristaltic pump into the microchip for 12 hr (72 ml total delivered volume).</li>
		<li>Remove the residual solution and then rinse the channel interior with D. I. H2O at a flow rate of 100 &#181;l min-1 via a peristaltic pump for 30 min (3 ml total delivered volume).</li>
		<li>Remove the residual D. I. H2O and then deliver a 0.5% (v/v) nitric acid (HNO3) solution into the microchip at a flow rate of 100 &#181;l min-1 via peristaltic pump for 30 min (3 ml total delivered volume).</li>
		<li>Remove the residual solution and then deliver a 50% (w/v) acrylamide solution into the microchip in the dark at a flow rate of 100 &#181;l min-1 via peristaltic pump for 8 hr (48 ml total delivered volume).</li>
		<li>Remove the residual solution and then rinse the channel interior with D. I. H2O at a flow rate of 100 &#181;l min-1 via a peristaltic pump for 30 min (3 ml total delivered volume).</li>
		<li>Pump air to remove the residual D. I. H2O with a peristaltic pump and then cover the microchip with an in-house-built photomask allowing the desired region of the extraction channel to be exposed to light. 
		Note: The in-house-built photomask was made of a black paper (114 mm (L) x 22 mm (W)) that contained an open window (94 mm (L) x 2 mm (W)) allowing the desired region of the extraction channel to be exposed to light.</li>
		<li>Preparation of the Cl-Containing SPE Formation Solution
		<ol>
			<li>Flush the inhibitor removal SPE cartridge with ethanol amounting to at least three cartridge-volumes.</li>
			<li>Flush the cartridge with 1,1&#39;-dichloroethene amounting to at least three cartridge-volumes prior to use.</li>
			<li>Pass 1 ml of 1,1&#39;-dichloroethene through the treated cartridge and then collect the fraction in a sample vial (20 ml) wrapped in aluminum foil.</li>
			<li>Add 491 &#956;l 1,1&#39;-dichloroethene into the solution containing 12 mg 2,2&#39;-azobisisobutyronitrile (AIBN), 3.18 ml ethanol, and 1.65 ml hexanes in 100-ml glass bottle.</li>
		</ol>
		</li>
		<li>Fill the chip channel with the Cl-containing SPE formation solution (approximately 200 &#956;l) by syringe injection and then expose the microchip to ultraviolet (UV365) irradiation with a maximum emission wavelength of 365 nm for 10 min (light intensity ~2.65 mW cm-2). 
		Caution! An appropriate exhaust system is recommended, because of the production of ozone during UV irradiation.</li>
		<li>Replace the residual solution with a fresh Cl-containing SPE formation solution (approximately 200 &#956;l) by syringe injection and then expose the microchip to UV365 irradiation for 10 min again (light intensity ~2.65 mW cm-2).</li>
		<li>Repeat Step 1.2.9 18 times.</li>
		<li>Rinse the channel interior with ethanol at flow rate of 100 &#181;l min-1 via peristaltic pump for 30 min (3 ml total delivered volume). After removing the residual solution with peristaltic pump, store the fabricated microchip in a zippered bag for the subsequent use.</li>
	</ol>
	</li>
</ol>
2. Surface Verification of PMMA Modification
<ol>
	<li>Contact Angle Analysis
	<ol>
		<li>Cut a PMMA sheet (350 mm (L) x 20 mm (W) x 2 mm (H)) into PMMA substrates (50 mm (L) x 20 mm (W) x 2 mm (H)) by the laser micromachining system.</li>
		<li>Immerse the PMMA substrates in 40 ml saturated NaOH solution in 50-ml conical tube and then agitate the resulting mixtures via a rocking shaker for 12 hr.</li>
		<li>Remove the residual solution and then rinse the PMMA substrates with 40 ml D. I. H2O.</li>
		<li>Immerse the PMMA substrates in 40 ml D. I. H2O and then agitate the resulting mixtures via a rocking shaker for 30 min.</li>
		<li>Remove the residual D. I. H2O. Immerse the PMMA substrates in a 40 ml of 0.5% (v/v) HNO3 solution and then agitate the resulting mixtures via a rocking shaker for 30 min.</li>
		<li>Remove the residual solution. Immerse the PMMA substrates in 40 ml of 50% (w/v) acrylamide solution and then agitate the resulting mixtures via a rocking shaker in the dark for 8 hr.</li>
		<li>Remove the residual solution and then rinse the PMMA substrates with 40 ml D. I. H2O.</li>
		<li>Immerse the PMMA substrates in 40 ml of D. I. H2O and then agitate the resulting mixtures via a rocking shaker for 30 min.</li>
		<li>Remove the residual D. I. H2O and then dry each PMMA substrate with a gentle stream of nitrogen for 2 min.</li>
		<li>Preparation of the Cl-Containing SPE Formation Solution
		<ol>
			<li>Flush the inhibitor removal SPE cartridge with ethanol amounting to at least three cartridge-volumes.</li>
			<li>Flush the cartridge with 1,1&#39;-dichloroethene amounting to at least three cartridge-volumes prior to use.</li>
			<li>Pass 6 ml of 1,1&#39;-dichloroethene through the treated cartridge and then collect the fraction in a sample vial (20 ml) wrapped in aluminum foil.</li>
			<li>Add 4.91 ml 1,1&#39;-dichloroethene into the solution containing 120 mg AIBN, 31.8 ml ethanol, and 16.5 ml hexanes in 100-ml glass bottle.</li>
		</ol>
		</li>
		<li>Apply 2 ml of the Cl-containing SPE formation solution onto the surfaces of the PMMA substrates and then expose the substrates to UV365 irradiation for 10 min (light intensity ~2.65 mW cm-2). 
		Caution! An appropriate exhaust system is recommended, because of the production of ozone during UV irradiation.</li>
		<li>Replace the residual solution with 2 ml of fresh Cl-containing SPE formation solution and then expose the substrates to UV365 irradiation for 10 min again (light intensity ~2.65 mW cm-2).</li>
		<li>Repeat Step 2.1.12 18 times.</li>
		<li>Remove the residual solution and then rinse the PMMA substrates with 40 ml ethanol in a 50-ml conical tube.</li>
		<li>Remove the residual solution and then rinse the PMMA substrates with 40 ml D. I. H2O.</li>
		<li>Remove the residual D. I. H2O and then dry each PMMA substrate with a gentle stream of nitrogen for 2 min.</li>
		<li>Drop 5 &#956;l D. I. H2O onto the PMMA substrates and determine the contact angle by a contact angle meter according to manufacturer&#39;s protocol. 
		Note: Use the average of three repeated measurements to determine the reported contact angles in each case.</li>
	</ol>
	</li>
	<li>Laser Ablation (LA)-Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) Analysis
	<ol>
		<li>Grind 8 g of PMMA beads into PMMA powders via a mortar and a pestle.</li>
		<li>Immerse the PMMA powders in 40 ml saturated NaOH solution in a 50-ml conical tube and then agitate the resulting mixtures via a rocking shaker for 12 hr.</li>
		<li>Remove the residual solution by a digital pipette with 5-ml tips and then rinse the PMMA powders with 40 ml D. I. H2O.</li>
		<li>Immerse the PMMA powders in 40 ml D. I. H2O and then agitate the resulting mixtures via a rocking shaker for 30 min.</li>
		<li>Remove the residual D. I. H2O. Immerse the PMMA powders in 40 ml of 0.5% (v/v) HNO3 solution and then agitate the resulting mixtures via a rocking shaker for 30 min.</li>
		<li>Remove the residual solution. Immerse the PMMA powders in 40 ml of 50% (w/v) acrylamide solution and then agitate the resulting mixtures via a rocking shaker in the dark for 8 hr.</li>
		<li>Remove the residual solution and then rinse the PMMA powders with 40 ml of D. I. H2O.</li>
		<li>Immerse the PMMA powders in 40 ml of D. I. H2O and then agitate the resulting mixtures via a rocking shaker for 30 min.</li>
		<li>Remove the residual D. I. H2O and then bake the PMMA powders at 60 &#176;C for 8 hr.</li>
		<li>Preparation of the Cl-Containing SPE Formation Solution
		<ol>
			<li>Flush the inhibitor removal SPE cartridge with ethanol amounting to at least three cartridge-volumes.</li>
			<li>Flush the cartridge with 1,1&#39;-dichloroethene amounting to at least three cartridge-volumes prior to use.</li>
			<li>Pass 16 ml 1,1&#39;-dichloroethene through the treated cartridge and then collect the fraction in a sample vial (20 ml) wrapped in aluminum foil.</li>
			<li>Add 14.73 ml 1,1&#39;-dichloroethene into the solution containing 360 mg AIBN, 95.4 ml ethanol, and 49.5 ml hexanes in a 250-ml glass bottle.</li>
		</ol>
		</li>
		<li>Mix the PMMA powders with 6 ml of Cl-containing SPE formation solution in a 50-ml conical tube, and equally transfer 1 ml of the mixture from conical tube into six wells of a 24-well tissue culture plate.</li>
		<li>Cover the tissue culture plate with a PMMA board and then expose the tissue culture plate to UV365 irradiation for 10 min (light intensity ~2.65 mW cm-2). 
		Caution! An appropriate exhaust system is recommended, because of the production of ozone during UV irradiation.</li>
		<li>Replace the residual solution with 1 ml of fresh Cl-containing SPE formation solution of each well and then expose the tissue culture plate to UV365 irradiation for 10 min again (light intensity ~2.65 mW cm-2).</li>
		<li>Repeat Step 2.2.13 18 times.</li>
		<li>Remove the residual solution and then rinse the PMMA powders in each well with 1 ml ethanol.</li>
		<li>Remove the residual solution and then rinse the PMMA powders in each well with 1 ml D. I. H2O.</li>
		<li>Remove the residual D. I. H2O and then bake the PMMA powders at 60 &#176;C for 8 hr.</li>
		<li>Compress the dried powders (1 g) to a pellet via a hydraulic press machine and then measure the signal for Cl by a LA-ICP-MS system. 
		Note: The signal for Cl at m/z 35 was selected as an indicator for the implanted C-Cl moieties. 
		A 193-nm laser was used as the ablation source. The Energy, Fluence, Spot Size, and Repetition Rate were set as 75%, 8.85 J cm-2, 100 &#181;m, and 5 Hz. At least 7 repeated measurements for each result were required. The LA-ICP-MS analytical procedures refer to published procedures elsewhere.20</li>
	</ol>
	</li>
	<li>Raman Spectroscopic Analysis
	<ol>
		<li>Perform the protocol from Step 2.2.1 to Step 2.2.17.</li>
		<li>Compress the dried powders (1 g) to a pellet via a hydraulic press machine and then take the spectra by a Raman spectrometer. 
		Note: Use a 780-nm laser line with maximum laser power of 100 mW as the photoexcitation source. Use the region of the Raman spectrum ranging from 550 to 900 cm-1 to investigate the attachment of the C-Cl moieties to the PMMA.</li>
	</ol>
	</li>
</ol>
3. Characterization of the Dipole-assisted SPE Reaction
<ol>
	<li>Perform the protocol from Step 2.2.1 to Step 2.2.17.</li>
	<li>Immerse 0.5 g PMMA powders in 5 ml of 20% (w/v) manganese nitrate tetrahydrate (Mn(NO3)24H2O) solution and then equally mix the resulting mixtures with 5 ml of 40 mM maleate buffer solution.</li>
	<li>Adjust the pH of the resulting mixtures to 8 by using a pure HNO3 solution and then agitate the mixtures via a rocking shaker for 1 hr.</li>
	<li>Remove the residual solution and then bake the PMMA powders at 60 &#176;C for 8 hr. Store the powders in 15-ml conical tube wrapped in aluminum foil for the X-ray absorption near-edge structure (XANES) analysis. 
	Note: The Mn K-edge XANES spectra were collected by using 07A and 17C1 beamlines of the National Synchrotron Radiation Research Center (NSRRC, Hsinchu, Taiwan). The electron storage ring was operated with energy of 1.5 GeV and a current of 100-200 mA. A Si(111) double crystal monochromator was used for providing highly monochromatized photon beams with energy of 1 to 15 keV and resolving power (E/&#916;E) of up to 5,000.The photon energy was calibrated by Mn standards using the known Mn K-edge absorption inflection point at 6539.0 eV. The Mn K-edge XANES spectra in the region between 6,530 and 6,570 eV were used to investigate the dipole-ion interactions for characterization of the proposed SPE reaction.</li>
</ol>

<ol>
	<li>De Mello, A. J., Beard, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dealing+with+'real'+samples:+sample+pre-treatment+in+microfluidic+systems.">Dealing with 'real' samples: sample pre-treatment in microfluidic systems.</a> Lab Chip. 3 (1), 11-19 (2003).</li><li>Lichtenberg, J., de Rooij, N. F., Verpoorte, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+pretreatment+on+microfabricated+devices.">Sample pretreatment on microfabricated devices.</a> Talanta. 56 (2), 233-266 (2002).</li><li>Song, S., Singh, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-chip+sample+preconcentration+for+integrated+microfluidic+analysis.">On-chip sample preconcentration for integrated microfluidic analysis.</a> Anal. Bioanal. Chem. 384 (1), 41-43 (2006).</li><li>Lafleur, J. P., Salin, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-concentration+of+trace+metals+on+centrifugal+microfluidic+discs+with+direct+determination+by+laser+ablation+inductively+coupled+plasma+mass+spectrometry.">Pre-concentration of trace metals on centrifugal microfluidic discs with direct determination by laser ablation inductively coupled plasma mass spectrometry.</a> J. Anal. At. Spectrom. 24 (11), 1511-1516 (2009).</li><li>Xue, S., Liu, Y., Li, H. -. F., Uchiyama, K., Lin, J. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microscale+solid-phase+extraction+poly(dimethylsiloxane)+chip+for+enrichment+and+fluorescent+detection+of+metal+ions.">A microscale solid-phase extraction poly(dimethylsiloxane) chip for enrichment and fluorescent detection of metal ions.</a> Talanta. 116, 1005-1009 (2013).</li><li>Khoai, D. V., Kitano, A., Yamamoto, T., Ukita, Y., Takamura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+high+sensitive+liquid+electrode+plasma-Atomic+emission+spectrometry+(LEP-AES)+integrated+with+solid+phase+pre-concentration.">Development of high sensitive liquid electrode plasma-Atomic emission spectrometry (LEP-AES) integrated with solid phase pre-concentration.</a> Microelectron. Eng. 111, 343-347 (2013).</li><li>Khoai, D. V., Yamamoto, T., Ukita, Y., Takamura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-chip+solid+phase+extraction-liquid+electrode+plasma+atomic+emission+spectrometry+for+detection+of+trace+lead.">On-chip solid phase extraction-liquid electrode plasma atomic emission spectrometry for detection of trace lead.</a> Jpn. J. Appl. Phys. 53 (5S1), 1-5 (2014).</li><li>Shih, T. -. T., Chen, W. -. Y., Sun, Y. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open-channel+chip-based+solid-phase+extraction+combined+with+inductively+coupled+plasma-mass+spectrometry+for+online+determination+of+trace+elements+in+volume-limited+saline+samples.">Open-channel chip-based solid-phase extraction combined with inductively coupled plasma-mass spectrometry for online determination of trace elements in volume-limited saline samples.</a> J. Chromatogr. A. 1218 (16), 2342-2348 (2011).</li><li>Chen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+solid+phase+microextraction+on+a+microchip+combined+with+electrothermal+vaporization-inductively+coupled+plasma+mass+spectrometry+for+determination+of+Cd,+Hg+and+Pb+in+cells.">Magnetic solid phase microextraction on a microchip combined with electrothermal vaporization-inductively coupled plasma mass spectrometry for determination of Cd, Hg and Pb in cells.</a> J. Anal. At. Spectrom. 25 (12), 1931-1938 (2010).</li><li>Kim, Y. H., Kim, G. Y., Lim, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro+Pre-concentration+and+Separation+of+Metal+Ions+Using+Microchip+Column+Packed+with+Magnetic+Particles+Immobilized+by+Aminobenzyl+Ethylenediaminetetraacetic+Acid.">Micro Pre-concentration and Separation of Metal Ions Using Microchip Column Packed with Magnetic Particles Immobilized by Aminobenzyl Ethylenediaminetetraacetic Acid.</a> Bull. Korean Chem. Soc. 31 (4), 905-909 (2010).</li><li>Strabburg, S., Wollenweber, D., Wunsch, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contamination+caused+by+ion-exchange+resin!?+Consequences+for+ultra-trace+analysis.">Contamination caused by ion-exchange resin!? Consequences for ultra-trace analysis.</a> Fresenius J. Anal. Chem. 360 (7-8), 792-794 (1998).</li><li>Watts, J. F., Chehimi, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+Photoelectron+Spectroscopy+Investigations+of+Acid-Base+Interactions+in+Adhesion.">X-ray Photoelectron Spectroscopy Investigations of Acid-Base Interactions in Adhesion.</a> J. Adhes. 41 (1-4), 81-91 (1993).</li><li>Eboatu, A. N., Diete-Spiff, S. T., Ezenweke, L. O., Omalu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Use+of+Polymers+as+Sequestering+Agents+for+Toxic+Metal+Ions.">The Use of Polymers as Sequestering Agents for Toxic Metal Ions.</a> J. Appl. Polym. Sci. 85 (13), 2781-2786 (2002).</li><li>Fiorini, G. S., Chiu, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disposable+microfluidic+devices:+fabrication,+function,+and+application.">Disposable microfluidic devices: fabrication, function, and application.</a> Biotechniques. 38 (3), 429-446 (2005).</li><li>Shadpour, H., Musyimi, H., Chen, J., Soper, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiochemical+properties+of+various+polymer+substrates+and+their+effects+on+microchip+electrophoresis+performance.">Physiochemical properties of various polymer substrates and their effects on microchip electrophoresis performance.</a> J. Chromatogr. A. 1111 (2), 238-251 (2006).</li><li>Shih, T. -. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dipole-assisted+solid-phase+extraction+microchip+combined+with+inductively+coupled+plasma-mass+spectrometry+for+online+determination+of+trace+heavy+metals+in+natural+water.">A dipole-assisted solid-phase extraction microchip combined with inductively coupled plasma-mass spectrometry for online determination of trace heavy metals in natural water.</a> Analyst. 140 (2), 600-608 (2015).</li><li>Yuan, D., Das, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+and+theoretical+analysis+of+direct-write+laser+micromachining+of+polymethyl+methacrylate+by+CO2+laser+ablation.">Experimental and theoretical analysis of direct-write laser micromachining of polymethyl methacrylate by CO2 laser ablation.</a> J. Appl. Phys. 101 (2), 1-6 (2007).</li><li>Xie, S., Svec, F., Frechet, J. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Porous+Hydrophilic+Monoliths:+Effect+of+the+Polymerization+Conditions+on+the+Porous+Properties+of+Poly(acrylamide-co-N,N'-methylenebisacrylamide)+Monolithic+Rods.">Preparation of Porous Hydrophilic Monoliths: Effect of the Polymerization Conditions on the Porous Properties of Poly(acrylamide-co-N,N'-methylenebisacrylamide) Monolithic Rods.</a> J. Polym. Sci. Pol. Chem. 35 (6), 1013-1021 (1997).</li><li>Ericson, C., Liao, J. -. L., Nakazato, K., Hjerten, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+continuous+beds+for+electrochromatography+and+reversed-phase+liquid+chromatography+of+low-molecular-mass+compounds.">Preparation of continuous beds for electrochromatography and reversed-phase liquid chromatography of low-molecular-mass compounds.</a> J. Chromatogr. A. 767 (1-2), 33-41 (1997).</li><li>Shih, T. -. T., Lin, C. -. H., Hsu, I. -. H., Chen, J. -. Y., Sun, Y. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Titanium+Dioxide-Coated+Microfluidic-Based+Photocatalyst-Assisted+Reduction+Device+to+Couple+High-Performance+Liquid+Chromatography+with+Inductively+Coupled+Plasma-Mass+Spectrometry+for+Determination+of+Inorganic+Selenium+Species.">Development of a Titanium Dioxide-Coated Microfluidic-Based Photocatalyst-Assisted Reduction Device to Couple High-Performance Liquid Chromatography with Inductively Coupled Plasma-Mass Spectrometry for Determination of Inorganic Selenium Species.</a> Anal. Chem. 85 (21), 10091-10098 (2013).</li><li>Carre, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polar+interactions+at+liquid/polymer+interfaces.">Polar interactions at liquid/polymer interfaces.</a> J. Adhes. Sci. Technol. 21 (10), 961-981 (2007).</li><li>Willis, H. A., Zichy, V. J. I., Hendra, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Laser-Raman+and+Infra-red+Spectra+of+Poly(Methyl+Methacrylate).">The Laser-Raman and Infra-red Spectra of Poly(Methyl Methacrylate).</a> Polymer. 10, 737-746 (1969).</li><li>Hendra, P. J., Mwkenzie, J. R., Holliday, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+laser-Raman+spectrum+of+polyvinylidene+chloride.">The laser-Raman spectrum of polyvinylidene chloride.</a> Spectroc. Acta Pt. A-Molec. Biomolec. Spectr. 25 (8), 1349-1354 (1969).</li></ol>

The authors have nothing to disclose.

The detailed procedures for the preparation of a dipole-assisted SPE microchip were presented above. In this section, the utility of the modification protocol with regard to the implantation of the C-Cl moieties on the PMMA and the feasibility of the Cl-containing PMMA, which was used as an extraction medium for the determination of trace metal ions, are evaluated step-by-step. For surface verification purposes, the sample type was selected on the basis of its compatibility with the analytical instrumentation. In other words, the types of test samples prepared via a similar process were determined in accordance with the requirements of the analytical instruments. For example, a substrate-type sample was used for the measurement of the contact angle, whereas a powder-packing-type sample was used for the LA-ICP-MS, Raman spectroscopic, and XANES analyses.
Initially, to monitor the change undergone by the chemical functionalities attached to the surface of the PMMA during the proposed procedures, a contact angle analysis for the resulting product corresponding to each step was carried out (Figure 2). As displayed in Figure 2, the variations in the contact angle clearly indicated that surface changes occurred during the modification procedures, and the contact angle of 80.3&#176; &#177; 0.43&#176; that was measured for the final product was in agreement with previously reported results.21
In addition, the existence of the C-Cl moieties on the modified PMMA was also confirmed via LA-ICP-MS analysis. Compared with the results obtained by ablating the native PMMA, distinct signals for Cl were observed expectably by ablating the PMMA modified with the C-Cl moieties (Figure 3(a)).
The Raman spectra were collected for further validating the attachment of the C-Cl moieties to the PMMA. As shown in Figure 3(b), two characteristic peaks associated with the CCl2 asymmetric stretching vibration were observed at 682 cm-1 and 718 cm-1 in the spectrum of the modified PMMA and that in reasonably good agreement with the results reported by Willis et al.22 and Hendra et al.23 In other words, the attachment of the C-Cl moieties to the PMMA could be successfully achieved after modification.
Moreover, to clarify the extraction mechanism proposed in this study, the XANES analysis was employed. As indicated in Figure 4, the interactions between the highly electronegative C-Cl moieties and the positively charged metal ions could be confirmed by the presence of the dominant absorption edge in the XANES spectrum corresponding to the modified PMMA treated with Mn2+ ions. Thus, the dipole-electrostatic interactions would be indeed applied to on-chip extraction for trace metal analyses. The detailed analytical results for water samples collected from two rivers in Taiwan have been described elsewhere.16
To the best of our knowledge, this is the first attempt to utilize an innovative working strategy in on-chip SPE reaction for the determination of trace metal ions, and that the developed device was significantly durable compared with other on-chip SPE techniques (i.e., more than 160 analytical works could be achieved without significant deterioration in terms of the extraction efficiency). Nevertheless, because such extraction mechanism was mainly relied on the interactions between the highly electronegative C-Cl moieties and the positively charged metal ions, the proposed technique was expected to be unsuited for the extraction of the negatively charged species so far.

From the viewpoints of environmental management and contamination prevention, trace metals which cause serious pollution or toxicological problems are a worldwide concern. An appropriate on-chip sample pretreatment technique has been widely accepted as the key to success in processing and analyzing actual samples via chip-based platforms, because unexpected co-existing chemical species in raw samples often hamper the accurate determination of analytes present in traces amounts.1 Among the available techniques, on-chip solid phase extraction (SPE) is especially popular for trace metal analyses, because this technique permitting sample cleanup and analyte preconcentration to be performed simultaneously is extremely useful for isolation of metal ions from complicated salt matrices.2,3
The advancement of on-chip SPE techniques used for the determination of trace metals has been steadily evolving. In the early days, the SPE chips were prepared by loading commercially available resins into the microchannels to construct the resin-packed SPE units.4-7 This occasionally required the analyte to be derivatized to enable the transformation of metal ions into resin-retainable forms.4 An alternative method for the preparation of chip-based SPE devices is to utilize the chip channel as an SPE sorbent for the collection of trace metals after simple surface modification.8 Recent years have seen an emerging trend involving the incorporation of magnetic nanoparticles (MNPs) and specific chemicals that contain functional groups capable of the efficient retention of metal ions. In contrast to commercial resins, the MNPs are modified with compounds such as &#947;-mercaptopropyltrimethoxysilane (&#947;-MPTS)9 and aminobenzyl ethylenediaminetetraacetic acid (ABEDTA)10 after which they are packed into the microchannels with the aid of an external magnetic field to achieve the selective extraction of metal ions.
Although significant progress in the development of on-chip SPE techniques has been witnessed, the reported techniques typically function based on either ion exchange or chelation. The use of techniques such as these has the disadvantage of requiring unavoidable operational procedures, including those associated with conditioning, washing, or regeneration, to maintain the analytical performance. Unfortunately, the need for additional operational procedures not only extends the time required for each analysis but also risks causing high blank values and irreproducible results.11 Therefore, an alternative working strategy for on-chip SPE techniques is imperative for trace metal analyses.
In 1993, Watts and Chehimi12 found that metal ions have a retention tendency toward polymeric materials, and that most of analytes efficiently retained on a chloro (Cl)-containing polymeric material, poly(vinyl chloride) (PVC) except sodium ions. Therefore, in 2002, Eboatu et al.13 further reported on the sequestration of some toxic metal from solutions by PVC. Because this indicated that Cl-containing polymeric materials exhibited superior properties for analyte preconcentration and salt matrix elimination, chip-based devices with the Cl-containing SPE functionality were considered an attractive strategy for the development of a novel on-chip SPE technique for the determination of trace metal ions. Considering material features, such as ease of fabrication, desired chemical/mechanical properties, and optical clarity,14,15 this study took advantage of poly(methyl methacrylate) (PMMA) to fabricate a microdevice. Then, the Cl-containing SPE functionality was implanted into the fabricated device for the development of a novel on-chip SPE technique for the determination of trace metal ions.16
Remarkably, the reliance of the innovative extraction mechanism on the dipole-ion interactions between the highly electronegative C-Cl moieties in the channel interior and the positively charged metal ions makes it possible to avoid measures taken during general on-chip SPE procedures, leading to a dramatic reduction of either the contamination caused by the use of excess reagents or the labor attributed to additional steps. The protocol provided in this contribution will enable researchers from diverse backgrounds to fabricate the dipole-assisted SPE microchip for their work. Detailed characterization procedures for the fabricated microchip are described as well.

<table border="0" cellpadding="0" cellspacing="0" width="1311">
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">AutoCAD</td>
			<td style="width:231px;">Autodesk</td>
			<td style="width:276px;">N/A</td>
			<td style="width:423px;">http://www.autodesk.com/education/free-software/autocad</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;">Poly(methyl methacrylate) (PMMA) sheet</td>
			<td style="width:231px;">Kun Quan Engineering Plastics</td>
			<td style="width:276px;">N/A</td>
			<td style="width:423px;">350 mm (L) x 20 mm (W) x 2 mm (H). The glass transition temperature (Tg) of PMMA sheets is ranged from 102&#8211;110 &#176;C. The UV transmittance of the PMMA at 365 nm is 91.2%.</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:383px;">Micromachining system</td>
			<td style="width:231px;">Laser Life</td>
			<td>LES-10</td>
			<td style="width:423px;">Maximum laser power: 10 W. Maximium engraving speed: 762 mm s&#8722;1.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">High-resolution optical microscope</td>
			<td style="width:231px;">Ching Hsing Computer-Tech</td>
			<td style="width:276px;">FS-230</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Power Image Analysis system (PIA)</td>
			<td style="width:231px;">Ching Hsing Computer-Tech</td>
			<td style="width:276px;">PIA V16.1</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Multi drilling machines</td>
			<td style="width:231px;">N/A</td>
			<td>LT-848</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Deionized water (D. I. H2O)</td>
			<td style="width:231px;">Millipore</td>
			<td>Milli-Q Integral 5 System</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Sodium dodecyl sulfate (SDS)</td>
			<td style="width:231px;">J. T. Baker</td>
			<td>4095-04</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Ultrasound oscillator</td>
			<td style="width:231px;">Elma</td>
			<td>Transsonic Digital</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Glass board</td>
			<td style="width:231px;">N/A</td>
			<td style="width:276px;">N/A</td>
			<td>160 mm (L) x 35 mm (W) x 2 mm (H); fragile</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;">Binder clip</td>
			<td style="width:231px;">SDI</td>
			<td style="width:276px;">0234T-1</td>
			<td style="width:423px;">http://stationery.sdi.com.tw/product_detail.php?Key=322&#38;cID=55&#38;uID=6</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Precision oven</td>
			<td style="width:231px;">Yeong Shin</td>
			<td style="width:276px;">DK-45</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">Poly(etheretherketone) (PEEK) tube</td>
			<td style="width:231px;">VICI</td>
			<td style="width:276px;">JR-T-6002 (0.5 mm i.d.); JR-T-6001 (0.25 mm i.d.)</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Polymer tubing&#160; cutter</td>
			<td style="width:231px;">Upchurch Scientific</td>
			<td style="width:276px;">A-327</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:53px;">Two-component epoxy-based adhesive</td>
			<td style="width:231px;">Richwang</td>
			<td style="width:276px;">N/A</td>
			<td style="width:423px;">Skin irritative. The major components are an epoxy resin and a hardener.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Peristaltic pump</td>
			<td style="width:231px;">Gilson</td>
			<td>Minipuls 3</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Peristaltic tube</td>
			<td style="width:231px;">Gilson</td>
			<td>F117934</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Sodium hydroxide (NaOH)</td>
			<td style="width:231px;">Sigma&#8211;Aldrich</td>
			<td style="width:276px;">30620</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Nitric acid (HNO3)</td>
			<td style="width:231px;">J. T. Baker</td>
			<td style="width:276px;">959834</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Acrylamide (prop-2-enamide, C3H5NO)</td>
			<td style="width:231px;">Sigma&#8211;Aldrich</td>
			<td style="width:276px;">A8887</td>
			<td>Acutely toxic and carcinogenic</td>
		</tr>
		<tr height="78">
			<td height="78" style="height:78px;width:383px;">In-house-built photomask</td>
			<td style="width:231px;">N/A</td>
			<td style="width:276px;">N/A</td>
			<td style="width:423px;">The in-house-built photomask was made of a black paper (114 mm (L) &#215; 22 mm (W)) that contained an open window (94 mm (L) &#215; 2 mm (W)) allowing the desired region</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">1,1-Dichloroethylene</td>
			<td style="width:231px;">Sigma&#8211;Aldrich</td>
			<td style="width:276px;">163032</td>
			<td>Acutely toxic and carcinogenic</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Cartridge</td>
			<td style="width:231px;">Dikma</td>
			<td style="width:276px;">ProElut AL-B&#160;</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">2,2-Azobisisobutyronitrile (AIBN, C8H12N4)</td>
			<td style="width:231px;">Showa Chemical&#160;</td>
			<td style="width:276px;">0159-2130</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Ethanol</td>
			<td style="width:231px;">Sigma&#8211;Aldrich</td>
			<td>32221</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Hexanes (C6H14)</td>
			<td style="width:231px;">Millinckrodt Chemical</td>
			<td style="width:276px;">5189-08</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:383px;">&#160;In-house-built irradiation system</td>
			<td style="width:231px;">Great Lighting (UV-A lamp)</td>
			<td style="width:276px;">N/A</td>
			<td style="width:423px;">An opaque box with an UV-A lamp (40 W, maximum emission at 365 nm)</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Glass vial</td>
			<td style="width:231px;">Yeong Shin</td>
			<td style="width:276px;">132300019</td>
			<td>Fragile</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Aluminum foil</td>
			<td style="width:231px;">Diamond</td>
			<td style="width:276px;">N/A</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">Conical tubes with screw caps</td>
			<td style="width:231px;">labcon</td>
			<td style="width:276px;">3181-345-008 (50 mL); 3131-345-008 (15 mL)</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Rocking shaker</td>
			<td style="width:231px;">TKS</td>
			<td style="width:276px;">RS-01</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Contact angle meter</td>
			<td style="width:231px;">First Ten Angstroms</td>
			<td>FTA 125</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">PMMA bead</td>
			<td style="width:231px;">Scientific Polymer Products</td>
			<td style="width:276px;">037A</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Mortar and pestle, agate&#160;</td>
			<td style="width:231px;">Yeong Shin</td>
			<td style="width:276px;">139000004</td>
			<td>Fragile</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">Tissue culture plate</td>
			<td style="width:231px;">AdvanGene Life Science Plasticware</td>
			<td style="width:276px;">AGC-CP-24S-50EA</td>
			<td>24-Well, non-treated, sterilized</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Hydraulic press</td>
			<td style="width:231px;">Panchum</td>
			<td style="width:276px;">Press-200</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Laser ablation</td>
			<td style="width:231px;">New Wave Research</td>
			<td>NWR193</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:26px;width:383px;">Inductively coupled plasma-mass spectrometer</td>
			<td style="width:231px;">Agilent Technologies</td>
			<td>Agilent 7500a</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:383px;">Glass bottle</td>
			<td style="width:231px;">DURAN</td>
			<td style="width:276px;">21801245 (100 mL); 21801365 (250 mL)</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Dispersive Raman spectrometer</td>
			<td style="width:231px;">Thermo Fisher Scientific</td>
			<td>Nicolet Almega XR</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Manganese nitrate tetrahydrate (Mn(NO3)2&#215;4H2O)</td>
			<td style="width:231px;">Sigma&#8211;Aldrich</td>
			<td style="width:276px;">63547</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:383px;">Maleic acid disodium salt hydrate (C4H4Na2O5)</td>
			<td style="width:231px;">Sigma&#8211;Aldrich</td>
			<td style="width:276px;">M9009</td>
			<td></td>
		</tr>
		<tr height="78">
			<td height="78" style="height:78px;width:383px;">X-ray absorption near edge structure (XANES)&#160;</td>
			<td style="width:231px;">N/A</td>
			<td style="width:276px;">N/A</td>
			<td style="width:423px;">The Mn K-edge XANES analyses were conducted at 07A and 17C1 beamlines of the National Synchrotron Radiation Research Center (NSRRC) in Taiwan.</td>
		</tr>
	</tbody>
</table>

fabrication of a dipole-assisted solid phase extraction microchip for trace metal analysis in water samples

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water samples. A brief overview of the evolution of chip-based SPE techniques is provided. This is followed by an introduction to specific polymeric materials and their role in SPE. To develop an innovative dipole-assisted SPE technique, a chlorine (Cl)-containing SPE functionality was implanted into a poly(methyl methacrylate) (PMMA) microchip. Herein, diverse analytical techniques including contact angle analysis, Raman spectroscopic analysis, and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) analysis were employed to validate the utility of the implantation protocol of the C-Cl moieties on the PMMA. The analytical results of the X-ray absorption near-edge structure (XANES) analysis also demonstrated the feasibility of the Cl-containing PMMA used as an extraction medium by virtue of the dipole-ion interactions between the highly electronegative C-Cl moieties and the positively charged metal ions.

Figure 2 depicts the reaction that occurs during the channel modification procedures of the PMMA microchip. Contact angle analysis was used to monitor the surface changes during the proposed procedures. An LA-ICP-MS system and a dispersive Raman spectrometer were employed to verify the successful modification of the C-Cl moieties formation on the PMMA substrate (Figure 3(a),(b)). The proposed dipole-assisted SPE reaction was characterized by the XANES analysis (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/53500/53500fig1.jpg" /> 
Figure 1. The PMMA microchip. (a) The snapshot of the pattern file for the fabricated microchip. (b) Layout of the fabricated microchip: S, E, and B represent the introduction ports for the sample, eluent, and buffer solutions, respectively; O represents the outlet. The black circle represents the drilled access hole for each. The channels used for the introduction of sample and buffer solutions both formed an angle of 30&#176; with the extraction channel. The length of the effective extraction channel, which was defined as the distance from the convergence point of the flows of the sample and buffer solutions to the confluent outlet, was 94 mm. (c) The photograph of the cross-section of the machined plate. Reproduced from Ref. 16 by permission of The Royal Society of Chemistry. <a href="https://www.jove.com/files/ftp_upload/53500/53500fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53500/53500fig2.jpg" /> 
Figure 2. Scheme of the channel modification for the PMMA microchip. The inset photographs show the contact angle corresponding to the resulting product in sequence. The contact angle was determined by using an image of a water drop. The average of three repeated measurements was used for determining the reported contact angles in each case. Reproduced from Ref. 16 by permission of The Royal Society of Chemistry. <a href="https://www.jove.com/files/ftp_upload/53500/53500fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53500/53500fig3.jpg" /> 
Figure 3. Surface verification of PMMA modification. (a) Signal for Cl obtained by ablating both the PMMA and PMMA modified with the C-Cl moieties. The inset shows the ablation positions corresponding to each obtaining signal. (b) Raman spectra of native and modified PMMA. Reproduced from Ref. 16 by permission of The Royal Society of Chemistry. <a href="https://www.jove.com/files/ftp_upload/53500/53500fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53500/53500fig4.jpg" /> 
Figure 4. Mn K-edge XANES spectra of modified PMMA and modified PMMA treated with Mn2+ ions. The spectra of modified PMMA was presented as red line. The interactions between the highly electronegative C-Cl moieties of modified PMMA and the Mn2+ ions shown the absorption spectra was presented as blue line. Reproduced from Ref. 16 by permission of The Royal Society of Chemistry. <a href="https://www.jove.com/files/ftp_upload/53500/53500fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Fabrication of a Dipole-assisted Solid Phase Extraction Microchip for Trace Metal Analysis in Water Samples

The fabrication protocol of a dipole-assisted solid phase extraction microchip for the trace metal analysis is presented.

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water ...

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8.6K Views.  National Tsing Hua University. The fabrication protocol of a dipole-assisted solid phase extraction microchip for the trace metal analysis is presented.

Video: Fabrication of a Dipole-assisted Solid Phase Extraction Microchip for Trace Metal Analysis in Water Samples

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and fold into higher order nanostructures. Structurally similar to peptides, peptoids are poly N-substituted glycines, where the side chains are attached to the nitrogen rather than the alpha-carbon. Their ease of synthesis and structural diversity allows testing of basic design principles to drive de novo design and engineering of new biologically-active and nanostructured materials.
Here, a simple manual peptoid synthesis protocol is presented that allows the synthesis of long chain polypeptoids ( up to 50mers) in excellent yields. Only basic equipment, simple techniques (e.g. liquid transfer, filtration), and commercially available reagents are required, making peptoids an accessible addition to many researchers' toolkits. The peptoid backbone is grown one monomer at a time via the submonomer method which consists of a two-step monomer addition cycle: acylation and displacement. First, bromoacetic acid activated in situ with N,N'-diisopropylcarbodiimide acylates a resin-bound secondary amine. Second, nucleophilic displacement of the bromide by a primary amine follows to introduce the side chain. The two-step cycle is iterated until the desired chain length is reached. The coupling efficiency of this two-step cycle routinely exceeds 98% and enables the synthesis of peptoids as long as 50 residues. Highly tunable, precise and chemically diverse sequences are achievable with the submonomer method as hundreds of readily available primary amines can be directly incorporated.
 
Peptoids are emerging as a versatile biomimetic material for nanobioscience research because of their synthetic flexibility, robustness, and ordering at the atomic level. The folding of a single-chain, amphiphilic, information-rich polypeptoid into a highly-ordered nanosheet was recently demonstrated. This peptoid is a 36-mer that consists of only three different commercially available monomers: hydrophobic, cationic and anionic. The hydrophobic phenylethyl side chains are buried in the nanosheet core whereas the ionic amine and carboxyl side chains align on the hydrophilic faces. The peptoid nanosheets serve as a potential platform for membrane mimetics, protein mimetics, device fabrication, and sensors. Methods for peptoid synthesis, sheet formation, and microscopy imaging are described and provide a simple method to enable future peptoid nanosheet designs.

A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the identification, localization, and characterization of nanoscale materials in biological samples is on the rise. Currently available methods, such as electron microscopy, tend to be resource-intensive, making their use prohibitive for much of the research community. Enhanced darkfield microscopy complemented with a hyperspectral imaging system may provide a solution to this bottleneck by enabling rapid and less expensive characterization of nanoparticles in histological samples. This method allows for high-contrast nanoscale imaging as well as nanomaterial identification. For this technique, histological tissue samples are prepared as they would be for light-based microscopy. First, positive control samples are analyzed to generate the reference spectra that will enable the detection of a material of interest in the sample. Negative controls without the material of interest are also analyzed in order to improve specificity (reduce false positives). Samples can then be imaged and analyzed using methods and software for hyperspectral microscopy or matched against these reference spectra in order to provide maps of the location of materials of interest in a sample. The technique is particularly well-suited for materials with highly unique reflectance spectra, such as noble metals, but is also applicable to other materials, such as semi-metallic oxides. This technique provides information that is difficult to acquire from histological samples without the use of electron microscopy techniques, which may provide higher sensitivity and resolution, but are vastly more resource-intensive and time-consuming than light microscopy.

Enhanced darkfield microscopy and hyperspectral imaging with spectral mapping enable screening, localization, and identification of nanoscale materials in histological samples with improved speed and accuracy over traditional methods. The goal of this paper is to provide methods for darkfield imaging and hyperspectral mapping of metal oxide nanoparticles in histological samples.

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the ...

Three-Dimensionally Printed Microfluidic Cross-flow System for Ultrafiltration/Nanofiltration Membrane Performance Testing

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane technology. Understanding the fouling process could lead to optimization and higher efficiency of membrane based filtration. Here we show the design and fabrication of an automated three-dimensionally (3-D) printed microfluidic cross-flow filtration system that can test up to 4 membranes in parallel. The microfluidic cells were printed using multi-material photopolymer 3-D printing technology, which used a transparent hard polymer for the microfluidic cell body and incorporated a thin rubber-like polymer layer, which prevents leakages during operation. The performance of ultrafiltration (UF), and nanofiltration (NF) membranes were tested and membrane fouling could be observed with a model foulant bovine serum albumin (BSA). Feed solutions containing BSA showed flux decline of the membrane. This protocol may be extended to measure fouling or biofouling with many other organic, inorganic or microbial containing solutions. The microfluidic design is especially advantageous for testing materials that are costly or only available in small quantities, for example polysaccharides, proteins, or lipids due to the small surface area of the membrane being tested. This modular system may also be easily expanded for high throughput testing of membranes.&#160;

Design and fabrication of a three-dimensionally (3-D) printed microfluidic cross-flow filtration system is demonstrated. The system is used to test performance and observe fouling of ultrafiltration and nanofiltration (thin film composite) membranes.

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane ...

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

Applications such as sensors, batteries, and fuel cells have been improved through the use of highly porous aerogels when functional compounds are encapsulated within the aerogels. However, few reports on encapsulating proteins within sol&#8211;gels that are processed to form aerogels exist. A procedure for encapsulating cytochrome c (cyt. c) in silica (SiO2) sol-gels that are supercritically processed to form bioaerogels with gas-phase activity for nitric oxide (NO) is presented. Cyt. c is added to a mixed silica sol under controlled protein concentration and buffer strength conditions. The sol mixture is then gelled and the liquid filling the gel pores is replaced through a series of solvent exchanges with liquid carbon dioxide. The carbon dioxide is brought to its critical point and vented off to form dry aerogels with cyt. c encapsulated inside. These bioaerogels are characterized with UV-visible spectroscopy and circular dichroism spectroscopy and can be used to detect the presence of gas-phase nitric oxide. The success of this procedure depends on regulating the cyt. c concentration and the buffer concentration and does not require other components such as metal nanoparticles. It may be possible to encapsulate other proteins using a similar approach making this procedure important for potential future bioanalytical device development.

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

This procedure describes how to encapsulate cytochrome c (cyt. c) in silica (SiO2) sol-gels, process these gels to form bioaerogels, and use these bioaerogels to rapidly recognize nitric oxide (NO) through a gas-phase reaction. This type of protocol may aid in the future development of biosensors or other bioanalytical devices.

Applications such as sensors, batteries, and fuel cells have been improved through the use of highly porous aerogels when functional compounds are ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...