micromachines

Article Fabricating Microstructures on for Microfluidic Chips by Glass Molding Process

Tao Wang 1, Jing Chen 1,*, Tianfeng Zhou 2,* and Lu Song 1

1 National Key Laboratory of Science and Technology on Micro/Nano Fabrication, Peking University, 100871, ; [email protected] (T.W.); [email protected] (L.S.) 2 Key Laboratory of Fundamental Science for Advanced Machining, Beijing Institute of Technology, Beijing 100081, China * Correspondence: [email protected] (J.C.); [email protected] (T.Z.); Tel.: +86-10-6726-6595 (J.C.); +86-10-6891-2716 (T.Z.)  Received: 28 April 2018; Accepted: 23 May 2018; Published: 29 May 2018 

Abstract: Compared with polymer-based biochips, such as polydimethylsiloxane (PDMS), glass based chips have drawn much attention due to their high transparency, chemical stability, and good biocompatibility. This paper investigated the glass molding process (GMP) for fabricating microstructures of microfluidic chips. The glass material was D-ZK3. Firstly, a mold with protrusion microstructure was prepared and used to fabricate grooves to evaluate the GMP performance in terms of roughness and height. Next, the molds for fabricating three typical microfluidic chips, for example, diffusion mixer chip, flow focusing chip, and cell counting chip, were prepared and used to mold microfluidic chips. The analysis of mold wear was then conducted by the comparison of mold morphology, before and after the GMP, which indicated that the mold was suitable for GMP. Finally, in order to verify the performance of the molded chips by the GMP, a mixed microfluidic chip was chosen to conduct an actual liquid filling experiment. The study indicated that the fabricating microstructure of glass microfluidic chip could be finished in 12 min with good surface quality, thus, providing a promising method for achieving mass production of glass microfluidic chips in the future.

Keywords: glass molding process; groove; roughness; filling ratio

1. Introduction Microfluidic devices have been drawing a great deal of attention among academic and engineering communities due to their potential to revolutionize analytical measurements in chemical and biomedical areas [1]. They are much smaller, lighter, and cheaper than traditional instruments, and can improve efficiency and reduce reagents’ consumption dramatically. Various microfluidic devices are fabricated for various applications, such as capillary electrophoresis [2,3], semen testing [4], electrochromatography [5], and DNA separation [6]. To date, many substrates have been reported to fabricate microfluidic chips, such as silicon [7], polydimethylsiloxane (PDMS) [8], polymethyl methacrylate (PMMA) [9], and glass [10]. Silicon has good chemical and thermal stability, and can obtain complicated 2D and 3D microstructure by photolithography and etching approaches, but the downsides of fragility, high-cost, opacity, poor electrical insulation, and complex surface chemical properties impede its application. Contrastingly, PDMS and PMMA are widely used, in both academic and industrial fields, for biochips due to their high efficiency and low cost of fabrication process. However, they are not appropriate for certain applications, such as operating hydrophobic molecules or when stable surface characteristics are required, due to the issues of dissolution and surface property control [11]. Among them, glass is proved to be a more suitable substrate for microfluidic chips, which can provide advantages over

Micromachines 2018, 9, 269; doi:10.3390/mi9060269 www.mdpi.com/journal/micromachines Micromachines 20182018,, 99,, 269x 22 of 1515

proved to be a more suitable substrate for microfluidic chips, which can provide advantages over other materials,materials, such as beneficialbeneficial opticaloptical properties,properties, goodgood insulatinginsulating properties,properties, high resistanceresistance toto mechanical stress,stress, highhigh surfacesurface stability,stability, and and high high solvent solvent compatibility compatibility of of the the glass glass [ 12[12].]. However, fabricating fabricating the the precise precise microstructure microstructure on glass on for glass microfluidic for microfluidic chips is still chips challenging is still andchallenging many researchers and many haveresearchers been exploring have been the exploring field of glassthe field microstructure of glass microstructure fabrication techniques.fabrication Generally,techniques. glass Generally, microstructure glass microstructure fabrication techniques fabrication can techniques be classified can intobe classified six categories, into six as categories, shown in Figureas shown1. in Figure 1.

Figure 1. SixSix typical typical glass glass microstructure microstructure fabrication fabrication techniques. techniques. (a) ( aWet) Wet etching; etching; (b) ( bDry) Dry etching; etching; (c) (Laserc) Laser fabrication; fabrication; (d) ( dMechanical) Mechanical fabrication; fabrication; (e) ( Photostructuring;e) Photostructuring; (f) (Moldingf) Molding process. process.

(1)(1) WetWet Etching Etching Glass is an isotropic material, which can be wet‐etched by buffered hydrofluoric acid (HF) in a Glass is an isotropic material, which can be wet-etched by buffered hydrofluoric acid (HF) non‐directional manner, as shown in Figure 1a. Wet‐etching technique is well developed in in a non-directional manner, as shown in Figure1a. Wet-etching technique is well developed in fabricating microfluidic channels on glass [13–16] and it can achieve the production of microfluidic fabricating microfluidic channels on glass [13–16] and it can achieve the production of microfluidic glass chips at a commercial scale at a reasonable cost. However, the wet‐etching process suffers some glass chips at a commercial scale at a reasonable cost. However, the wet-etching process suffers inherent limitations, such as undercut, which exerts a challenge to fabricate high aspect ratio some inherent limitations, such as undercut, which exerts a challenge to fabricate high aspect ratio microstructure. In addition, the chemical liquid used in the process, especially buffered HF, is microstructure. In addition, the chemical liquid used in the process, especially buffered HF, is harmful harmful to the environment, which needs further disposal before discharging. to the environment, which needs further disposal before discharging. (2) Dry Etching (2) Dry Etching Dry chemical etching of glass is carried out by capacitively coupled plasmas (CCP) [17], Dry chemical etching of glass is carried out by capacitively coupled plasmas (CCP) [17], CCP/microwave plasma [18] and inductively coupled plasmas (ICP) [19]. It can achieve an CCP/microwave plasma [18] and inductively coupled plasmas (ICP) [19]. It can achieve an anisotropic anisotropic and precise profile, as shown in Figure 1b. Reactive ion etching results in an anisotropic and precise profile, as shown in Figure1b. Reactive ion etching results in an anisotropic profile due to profile due to the directional nature of the ion bombardment, affecting surface chemical reaction, as the directional nature of the ion bombardment, affecting surface chemical reaction, as well as physical well as physical sputtering [20]. The technique for fabricating glass has been reported using different sputtering [20]. The technique for fabricating glass has been reported using different chemistries, chemistries, such as C3F8, CHF3, CF4/CHF3, SF6/Ar and CF4/Ar [17–21]. The major weaknesses of glass such as C3F8, CHF3, CF4/CHF3, SF6/Ar and CF4/Ar [17–21]. The major weaknesses of glass dry dry etching are the low etch rate and the low etching selectivity of the glass to the etch mask, which etching are the low etch rate and the low etching selectivity of the glass to the etch mask, which impede impede its widespread application substantially. its widespread application substantially.

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(3) Laser Fabrication

Laser fabrication of microstructure on glass has been reported using CO2, UV, and ultra-short pulse lasers [22–24], and its mechanism is shown in Figure1c. Although the efficiency of laser glass micromachining is much higher than those of other conventional methods, its wide application is hindered by the brittleness and poor thermal properties of glass, resulting in a risk of microcracking and poor surface quality on the bottom of groove [25]. (4) Mechanical Fabrication The major mechanical fabrication techniques include micromilling [26,27], powder blasting [28–30], and micro-ultra-sonic machining [31]. They are superior in fabricating efficiency, despite it usually being a challenge to obtain smooth machined surfaces by mechanical fabrication. In addition, the minimum size of the microstructure mainly depends on the tool. (5) Photostructuring Some photosensitive glass is commercially available, with the material itself being sensitive to ultraviolet (UV) light of a wavelength of around 310 nm (e.g., Schott Fortran [32]), which is amenable to anisotropic photostructuring. Therefore, it does not require an intermediate photoresist layer for patterning, as shown in Figure1e. Although the fabrication process is simpler than the conventional wet-etching process, the whole processing period is still long at over 20 h. This is due to the required heating and cooling processes. In addition, the cost of Schott Fortran glass is greater than for conventional glass. (6) Molding Process Glass is a strongly temperature-dependent material. It is hard and brittle at room temperature, while it becomes a viscoelastic body or viscous liquid at high temperate. To date, the GMP has been accepted as a promising technique to efficiently generate microstructure on glass [33–35], as shown in Figure1f. The main challenges are the microstructure fabrication on mold and the optimization of parameters for GMP. Although GMP has been investigated in fabricating microstructure on glass for decades, the majority of the published reports focus on optical components [33–38], in which the typical microstructures are arrays of microgrooves, micropyramids, microneedles, microlenses, and microprisms. However, in reference to fabricating microfluidic chips, the typical microstructure is a U or rectangle cross-section groove. Recently, some researchers have started to shift the focus to the fabrication of microfluidic chips. Chen et al. [39,40] used nickel alloy mold to conduct their GMP for fabricating microfluidic chips in a conventional furnace, instead of a large-volume vacuum hot press. Since there is no water cooling system, the entirety of the GMP lasted more than 15 h. Huang et al. [41] utilized silicon molds to conduct the GMP in a GMP-207-HV (Toshiba Machine Co., Ltd., Shizuoka-ken, Japan) optical glass mold press machine for fabricating microfluidic chips, and the time of the GMP could, therefore, be reduced to less than 15 min. Since silicon material is inherently brittle, it is easy for fractures to occur during the GMP. This paper explored the microstructure fabrication method of GMP on glass for microfluidic chips, which could lay an experimental and theoretical foundation for achieving mass production of microfluidic glass chips in the future. Since the glass–glass bonding process is an important issue for glass microfluidic chips, it will be investigated thoroughly in the future. Therefore, the chip verification in this paper was only conducted on a tape-glass bonding chip for simplification. In the present research, groove molding experiments were conducted and the molded profiles were observed. Moreover, the influence of the molding parameters was investigated, with corresponding experiments investigating the molding of microfluidic chips also being conducted and the molded chips were demonstrated. Finally, issues surrounding the curved side wall and bonding technique were discussed. Micromachines 2018, 9, 269 4 of 15 Micromachines 2018, 9, x 4 of 15 Micromachines 2018, 9, x 4 of 15 2. Experiments 2. Experiments 2.1. Experimental Setup and Measurement 2.1. Experimental Setup and Measurement All experiments experiments were were carried carried out out on on an anultraprecision ultraprecision glass glass molding molding machine, machine, PFLF7 PFLF7-60A‐60A (SYS All experiments were carried out on an ultraprecision glass molding◦ machine, PFLF7‐60A (SYS (SYSCorp., Corp., Tochigi, Tochigi, Japan), Japan), which which could could operate operate between between 20–750 20–750 °C TheC The glass glass molding molding process process is Corp., Tochigi, Japan), which could operate between 20–750 °C The glass molding process is schematicallyis schematically illustrated illustrated in in Figure Figure 2.2 .The The GMP GMP consists consists of of four four steps: Heating, pressing, annealing, schematically illustrated in Figure 2. The GMP consists of four steps: Heating, pressing, annealing, and cooling. In the first first step, the glass and mold are placed on the bottom platen and heated together and cooling. In the first step, the glass and mold are placed on the ◦bottom platen and heated together until the molding temperature is reached, which is typically 10 °CC above the transition temperature until the molding temperature is reached, which is typically 10 °C above the transition temperature of the glass material, as shown in Figure2 2a.a. Next,Next, thethe upperupper platenplaten isis drivendriven downwarddownward toto conductconduct of the glass material, as shown in Figure 2a. Next, the upper platen is driven downward to conduct the pressing process, which achieves the replication of the microstructure from the mold onto the the pressing process, which achieves the replication of the microstructure from the mold onto the preform. Then, the temperature is slowly cooled down to somewhat below the annealing point, while preform. Then, the temperature is slowly cooled down to somewhat below the annealing point, while a small pressing pressure is used to maintain high fidelity, fidelity, as shown in Figure 22c.c. Finally, thethe preformpreform a small pressing pressure is used to maintain high fidelity, as shown in Figure 2c. Finally, the preform experiences the fast cooling step via the water cooling system and the microstructure is obtained by experiences the fast cooling step via the water cooling system and the microstructure is obtained by demolding, asas shownshown in in Figure Figure2d. 2d. The The typical typical evolution evolution of temperature of temperature and pressureand pressure during during the GMP the demolding, as shown in Figure 2d. The typical evolution of temperature and pressure during the GMPin experiment in experiment is shown is shown in Figure in Figure3. 3. GMP in experiment is shown in Figure 3.

Figure 2. The schematic photo of the GMP. (a) Heating; (b) Pressing; (c) Annealing; (d) Cooling FigureFigure 2. 2.TheThe schematic schematic photo photo of ofthe the GMP. GMP. (a ()a Heating;) Heating; (b ()b Pressing;) Pressing; (c ()c Annealing;) Annealing; (d ()d Cooling) Cooling.

Figure 3. The typical evolution of temperature and pressure during the glass molding process (GMP). FigureFigure 3. 3.TheThe typical typical evolution evolution of oftemperature temperature and and pressure pressure during during the the glass glass molding molding process process (GMP). (GMP). The surface morphology of the mold was observed by a VK‐X200 3D Laser Scanning Microscope The surface morphology of the mold was observed by a VK‐X200 3D Laser Scanning Microscope (KeyenceThe surface Corporation, morphology Osaka, of Japan) the mold and was FEI observedQuanta 250 by aFEG VK-X200 (Thermo 3D Fisher Laser Scanning Scientific, Microscope Waltham, (Keyence Corporation, Osaka, Japan) and FEI Quanta 250 FEG (Thermo Fisher Scientific, Waltham, (KeyenceMA, USA). Corporation, The surface Osaka,morphology Japan) of and the FEImolded Quanta glass 250 was FEG studied (Thermo by an Fisher Olympus Scientific, Lext Waltham,OLS4100 MA, USA). The surface morphology of the molded glass was studied by an Olympus Lext OLS4100 (OlympusMA, USA). Corporation, The surface morphologyTokyo, Japan) of and the moldedall the roughness glass was measurements studied by an Olympuswere conducted Lext OLS4100 by this (Olympus Corporation, Tokyo, Japan) and all the roughness measurements were conducted by this (Olympusas well. Corporation, Tokyo, Japan) and all the roughness measurements were conducted by this as well. as well. 2.2. Glass and Mold 2.2.2.2. Glass Glass and and Mold Mold The D‐ZK3 optical glass wafer, with a 25.4 mm diameter and 0.6 mm thickness, was used in the The D‐ZK3 optical glass wafer, with a 25.4 mm diameter and 0.6 mm thickness, was used in the experiment.The D-ZK3 The opticalmaterial glass is dense wafer, barium with a crown 25.4 mm optical diameter glass and manufactured 0.6 mm thickness, by CDGM was GLASS used in Co., the experiment. The material is dense barium crown optical glass manufactured by CDGM GLASS Co., LTDexperiment. (Chengdu, The China). material The is densemajor chemical barium crown compositions optical glass and thermal manufactured properties by CDGM are listed GLASS in Tables Co., LTD (Chengdu, China). The major chemical compositions and thermal properties are listed in Tables 1LTD and (Chengdu, 2, respectively. China). Tg Theis defined major chemicalas the transition compositions temperature, and thermal above properties which the are volume listed expanding in Tables1 1 and 2, respectively. Tg is defined as the transition temperature, above which the volume expanding rateand 2increases, respectively. abruptlyTg is as defined the temperature as the transition increases. temperature, Ts is defined above as which the yielding the volume temperature, expanding at rate increases abruptly as the temperature increases. Ts is defined as the yielding temperature, at whichrate increases the glass abruptly reaches as its the maximum temperature expansion increases. pointTs is and defined starts as shrinking the yielding as temperature temperature, increases at which which the glass reaches its maximum expansion point and starts shrinking as temperature increases further.the glass In reaches order itsto shorten maximum the expansion paper length, point the and morphology starts shrinking details as of temperature molds are increaseslisted in Figures further. further. In order to shorten the paper length, the morphology details of molds are listed in Figures S1–S5 in Supplementary Materials. S1–S5 in Supplementary Materials.

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In order to shorten the paper length, the morphology details of molds are listed in Figures S1–S5 in Supplementary Materials.

Table 1. The major thermal properties of D-ZK3 glass [42].

◦ ◦ 14.5 ◦ 13 ◦ 7.6 ◦ −7 Content Tg ( C) Ts ( C) T10 ( C) T10 ( C) T10 ( C) α100/300 ◦C (10 /K) Value 511 546 471 499 605 93

Table 2. The chemical compositions of D-ZK3 glass [42].

Content SiO2 B2O3 CaO Al2O3 BaO Sb2O3 Value 30–40% 20–30% 0–10% 0–10% 10–20% 0–10%

2.3. Design of Experiment The main factors influencing the molded morphology are the pressing temperature, pressure, and ◦ time. The pressing temperature is usually set 10 C above the transition temperature, Tg, according to previous experiments. In this paper, the pressing temperature range was set between 547 ◦C and 552 ◦C, which means that the adhesion between the glass and the mold was avoided and demonstrates the influence of temperature. In regards to pressure, this was set between 0.1 MPa and 0.5 MPa, which is the main molding pressure in the molding machining. Pressing times was set as 80s in all experiments as its influence is insignificant if the time is above 60 s, based on our previous research. The present experiment consisted of two parts. The first part was the fundamental study investigating the influence of molding parameters, such as temperature and pressure, on the morphology of the molded groove, which can help to ascertain the optimal parameters. The second part was the investigation of the microstructure morphology during the fabricating of microfluidic chips by the GMP. The details of the parameters in the experiment design are listed in Table3.

Table 3. Details of the parameters in the glass molding process (GMP).

Item No. Temperature (◦C) Pressure (MPa) 1 547 0.3 2 548 0.3 3 549 0.3 Varying temperature 4 550 0.3 5 551 0.3 6 552 0.3 7 549 0.1 8 549 0.2 Varying pressure 9 549 0.4 10 549 0.5 Diffusion mixer chip 11 550 0.5 Flow focusing chip 12 550 0.5 Cell counting chip 13 550 0.5

3. Results and Discussion

3.1. Groove Molding Experiment The aim of this study was to investigate molding performance during the fabricating of grooves with a 60 µm depth. The molded grooves at different molding temperatures are shown in Figure4. There are three grooves with a width of 200 µm, 100 µm, and 50 µm, respectively in each photo. All bottom surfaces of the grooves are extremely smooth. In order to get the quantitative value of the Micromachines 2018, 9, 269 6 of 15

Micromachines 2018, 9, x 6 of 15 surface quality at the groove bottom, the sample molded at the temperature of 547 ◦C was taken and the surfacebetween roughness the channel measurementswidth and the mold were surface taken roughness, of all its bottom value is surfaces mainly determined running by parallel the to the mold surface roughness due to the contact between the mold surface and the groove bottom surface grooves by the Olympus Lext OLS4100 (Olympus Corporation, Tokyo, Japan), as shown in Figure4a. during the molding process. Due to the polishing process, the surface roughness, Ra, of the mold can The resultsachieve demonstrate around 10 that nm. the However, surface as roughness, an exploring Rinvestigation,a, with the the width molding of 200 experimentµm, 100 wasµm, not and 50 µm, are 13 nm,conducted 9 nm, and in 7a nm,clean respectively.room and the individual Although roughness the results value somewhat may be influenced indicate by asome correlation dust on between the channelthe width groove and bottom the surface, mold surfaceleading to roughness, small variations its valuein the measurements is mainly determined at different locations. by the mold surface It is worth mentioning that the bottom of the groove fabricated by the GMP is much better than roughnessthose due of to the the conventional contact between fabricating the methods, mold which surface is beneficial and the to groove the reduction bottom of residual surface bio during‐ the molding process.samples and Due the to increase the polishing of light process,transmittance the performance. surface roughness, In addition,Ra ,when of the temperature mold can achieve around 10increases nm. However, to 551 °C, as some an exploring rough zones investigation, can be observed the between molding grooves, experiment and as temperature was not conducted in a clean roomincreases and further, the individualthe size of the roughness zones increase value correspondingly. may be influenced Since the surface by some roughness dust of on the the groove mold during the GMP is relatively rough (Ra around 0.3 μm) due to its fabricating process, it is bottom surface,expected leading that this to zone small touched variations the bottom in of the the measurements mold during the GMP. at different locations.

Figure 4. TheFigure molded 4. The molded grooves grooves at different at different molding molding temperatures (P = ( P0.3= MPa). 0.3 MPa). (a) 547 (°C;a) ( 547b) 548◦C; °C; ( b) 548 ◦C; (c) 549 °C; (d) 550 °C; (e) 551 °C; (f) 552 °C. (c) 549 ◦C; (d) 550 ◦C; (e) 551 ◦C; (f) 552 ◦C. In order to provide more information about the molded grooves, the profiles perpendicular to It is worththe grooves mentioning were extracted that in the each bottom sample, as of shown the groove in Figure fabricated 5. It is evident by that the the GMP depth isof muchthe better groove increases with the molding temperature, from 40 μm at 547 °C to 60 μm at 550 °C. The corner than thoseradius of the of conventional the grooves decreases fabricating with methods,the molding which temperature is beneficial also. In to addition, the reduction when the of residual bio-samplestemperature and the reaches increase 550 °C, of the light glass transmittance almost touches the performance. mold bottom and In when addition, it increases when further, temperature increases tothe 551 contact◦C, somezones in rough the profiles zones can can be be observed observed as the between rough and grooves, horizontal and areas as temperatureon the top, as increases further, theshown size ofin Figure the zones 5e,f. increase correspondingly. Since the surface roughness of the mold during The influence of the pressure is similar to that of the temperature. In order to avoid redundancy, µ the GMP isthe relatively microstructure rough images (Ra andaround profiles 0.3 moldedm) due at different to its fabricatingpressures are process,ignored in itthe is paper. expected The that this zone touchedinfluence the bottomof the molding of the parameters, mold during such as the pressure GMP. and temperature, on groove depth are shown In orderin Figures to provide 6 and 7, more respectively. information It indicates about that the the molded groove depth grooves, increased the profileswith both perpendicular parameters. to the grooves wereThe depth extracted value inreached each was sample, maximum as shown at a pressure in Figure of 0.4 5MPa,. It is with evident the temperature that the set depth at 549 of °C, the groove and at 550 °C when the pressure was set at 0.3 MPa. It is worth noting that, although further increases µ ◦ µ ◦ increases withof both the parameters molding would temperature, benefit the from fill ratio, 40 mthis at increases 547 C tothe 60 risk mof atcracking 550 C.in Thethe glass, corner in radius of the groovesaddition decreases to adhesion with thebetween molding the mold temperature and the glass. also. Therefore, In addition, the investigation when the of temperature the optimal reaches 550 ◦C, theparameters glass almost for the touches maximum the filling mold ratio bottom are not included and when in the it scope increases of the paper. further, Nevertheless, the contact all zones in the profiles can be observed as the rough and horizontal areas on the top, as shown in Figure5e,f. The influence of the pressure is similar to that of the temperature. In order to avoid redundancy, the microstructure images and profiles molded at different pressures are ignored in the paper. The influence of the molding parameters, such as pressure and temperature, on groove depth are shown in Figures6 and7, respectively. It indicates that the groove depth increased with both parameters. The depth value reached was maximum at a pressure of 0.4 MPa, with the temperature set at 549 ◦C, and at 550 ◦C when the pressure was set at 0.3 MPa. It is worth noting that, although further increases Micromachines 2018, 9, x 7 of 15 the parameters of temperature and pressure used in this experiment were sufficient to avoid adhesion Micromachines 2018, 9, 269 7 of 15 during the demolding process. The phenomenon of typical broken up in glass after demolding is shown in Figure 8. The whole part of the bottom material breaks away from the top material, which shouldof both be parameters avoided in would the GMP. benefit the fill ratio, this increases the risk of cracking in the glass, in addition to adhesion between the mold and the glass. Therefore, the investigation of the optimal parameters for the maximumMicromachines filling 2018 ratio, 9, x are not included in the scope of the paper. Nevertheless, all the7 of parameters 15 of temperature and pressure used in this experiment were sufficient to avoid adhesion during the demoldingthe parameters process. Theof temperature phenomenon and pressure of typical used in broken this experiment up in were glass sufficient after demolding to avoid adhesion is shown in during the demolding process. The phenomenon of typical broken up in glass after demolding is Figure8. (μm) Height The whole part of the bottom material breaks away from the top material, which should be shown in Figure 8. The whole part of the bottom material breaks away from the top material, which avoided inshould the GMP.be avoided in the GMP. Height (μm) Height Height (μm) Height (μm) Height (μm) Height Height (μm) Height Height (μm) Height Height (μm) Height

Figure 5. The profiles of the molded grooves at different molding temperatures. (a) 547 °C; (b) 548 °C; FigureFigure 5. 5. TheThe profiles profiles of of the the molded molded grooves grooves at at different different molding molding temperatures. temperatures. ( (aa)) 547 547 °C;◦C; (b (b)) 548 548 °C;◦C; (c) 549 °C; (d) 550 °C; (e) 551 °C; (f) 552 °C. ((cc)) 549 549 °C;◦C; (d (d)) 550 550 °C;◦C; (e (e) )551 551 °C;◦C; (f ()f )552 552 °C.◦C.

Figure 6. The influence of pressure on depth.

FigureFigure 6. 6. TheThe influence influence of of pressure pressure on on depth. depth.

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Micromachines 2018, 9, 269 8 of 15

Micromachines 2018, 9, x 8 of 15 Micromachines 2018, 9, x 8 of 15

Figure 7. The influence of temperature on depth.

FigureFigure 7. 7. TheThe influence influence of of temperature temperature on on depth. depth. Figure 7. The influence of temperature on depth.

Figure 8.8. A A broken broken glass. glass. FigureFigure 8. 8. AA broken broken glass. glass. In orderIn order to toreveal reveal the the accuracy accuracy ofof thethe molded glass glass grooves grooves relative relative to the to theactual actual mold mold features, features, InIn order order to to reveal reveal the the accuracy accuracy of of the the molded molded glass glass grooves grooves relative relative to to the the actual actual mold mold features, features, the thewidth width comparisons comparisons between between moldmold protrusions and and glass glass grooves grooves are areshown shown in Figure in Figure 9. The 9. The samplethethe width of comparisonscomparisonsglass groove between betweendata is mold extractedmold protrusions protrusions from the and andprofile glass glass grooves at groovesa temperature are shown are shown in of Figure 552in Figure9°C,. The which sample9. The is sample of glass groove data is extracted from the profile at a temperature◦ of 552 °C, which is demonstratedsampleof glass of groove glass at data thegroove bottom is extracted data of Figureis fromextracted 5. the It indicates profile from the at that a profile temperature the widths at a temperature of 552moldC, protrusions which of 552 is demonstrated °C,are allwhich wider is demonstrated at the bottom of Figure 5. It indicates that the widths of mold protrusions are all wider thandemonstratedat the the bottom corresponding at of the Figure bottom widths5. of It Figure indicatesof glass 5. grooves, It that indicates the and widths that that the the of widths molding mold of protrusions moldaccuracy protrusions was are around all are wider all 4–6 wider than μm. thanThethanthe the corresponding differentthecorresponding corresponding widths widths widthscan widths be of of glass attributedof glass glass grooves, grooves,grooves, to the and and andfact that that thatthat, the the the moldingwhen molding molding the accuracy accuracy force accuracy from waswas was thearound protrusionsaround 4–6 4–6 μ µ4–6m.m. μm. Thecompressing TheThedifferent differentdifferent widths the widths widths glass cancan preformcan be be attributedbe attributed attributedis taken to off the to by fact the thedemolding, that, factfact when that,that, thethe when forcewhenwalls the from of the theforce the forceglass protrusions from groovesfrom the compressingtheprotrusions experience protrusions compressingspringback,compressingthe glass preformthe therefore, the glass glass is preform taken leadingpreform off tois by is thetaken taken demolding, smaller off by glass the demolding,demolding, groove walls of width. the the walls glass walls groovesof theof the glass experience glass grooves grooves springback,experience experience springback,springback,therefore, therefore, leading therefore, to leading the leading smaller to to the glassthe smallersmaller groove glassglass width. groove groove width. width. 220 220 192 220200 186 180200 192 186 200 Mold protrusion 192 160180 Mold protrusion 186 180 Glass groove 140160 GlassMold groove protrusion 160120140 Glass groove 140100120 93 89 12010080 93 89 93 Width100 (μm) 6080 47 41 89 Width (μm)804060 47 41 40 Width (μm) 6020 47 41 40200 0 123 20 123Test No. 0 123Test No. Figure 9. The width comparison between mold protrusions and glass grooves. Figure 9. The width comparison betweenTest mold No. protrusions and glass grooves. 3.2. Microfluidic Chips 3.2. MicrofluidicFigureFigure Chips 9. The 9. The width width comparison comparison between between mold mold protrusions protrusions and and glass glass grooves. grooves. A typical microstructure of a molded diffusion mixer chip is shown in Figure 10. The macro 3.2. photoMicrofluidicA indicatestypical Chips microstructure that the basic ofmorphology a molded diffusionof the diffusion mixer chip mixer is shownchip was in achieved.Figure 10. InThe order macro to providephoto indicates more details that theof thebasic molded morphology chip, area of theA (which diffusion is themixer combination chip was areaachieved. of the In two order input to provideA typical more microstructure details of the moldedof a molded chip, area diffusion A (which mixer is the chip combination is shown areain Figure of the 10.two Theinput macro photo indicates that the basic morphology of the diffusion mixer chip was achieved. In order to provide more details of the molded chip, area A (which is the combination area of the two input

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3.2. Microfluidic Chips A typical microstructure of a molded diffusion mixer chip is shown in Figure 10. The macro photo indicates that the basic morphology of the diffusion mixer chip was achieved. In order to provide more details of the molded chip, area A (which is the combination area of the two input channels) Micromachines 2018, 9, x 9 of 15 and area B (which is in the typical U-shape mixed area) were enlarged, as is shown in Figure 10b,c, respectively.channels) Since and it isarea a small B (which area, is in the the highest typical U depth‐shape mixed was only area) 23.9were µenlarged,m in Figure as is shown 10b. Thein Y shape was formed,FigureMicromachines which 10b,c, 2018 is respectively. suitable, 9, x forSince the it is two a small liquids area, the combining. highest depth In was order only to 23.9 provide μm in Figure more9 10b. of details 15 of the The Y shape was formed, which is suitable for the two liquids combining. In order to provide more morphology, the profile extraction was conducted along the location, as shown in Figure 10b, and the detailschannels) of the and morphology, area B (which the profileis in the extraction typical Uwas‐shape conducted mixed alongarea) thewere location, enlarged, as shown as is shownin Figure in profile is shown10b,Figure and 10b,c, inthe Figure profilerespectively. is 10 shownd. Since Although in Figureit is a small 10d. the Althougharea, corner the highest radiusthe corner depth is radius relatively was onlyis relatively 23.9 large, μm large, in it Figure can it can be 10b. be reduced by further parameterreducedThe Y shape by optimization. further was formed, parameter which In optimization. addition, is suitable for it In is theaddition, also two valuable liquids it is also combining. ifvaluable there In areif orderthere some toare provide roundsome round more corners in the cross-sectioncornersdetails profile of in the the morphology, of cross the‐section channel the profile profile in microfluidicof extractionthe channel was in chips. conductedmicrofluidic Regarding along chips. the Regarding thelocation, mixing as the shown zone,mixing in Figure this zone, profile was this profile was also extracted, and is shown in Figure 10e. It indicates that the channels running also extracted,10b, and and the is profile shown is shown in Figure in Figure 10e. 10d. It indicates Although thatthe corner the channels radius is relatively running large, parallel it can to be each other, parallelreduced to by each further other, parameter with a depth optimization. of 40μm, Incan addition, be formed it is by also the valuable GMP, which if there is deep are some enough round for µ with a depththecorners mixing of 40 in thefunction.m, cross can‐ section be formed profile by of the the channel GMP, in which microfluidic is deep chips. enough Regarding for thethe mixing zone, function. this profile was also extracted, and is shown in Figure 10e. It indicates that the channels running parallel to each other, with a depth of 40μm, can be formed by the GMP, which is deep enough for the mixing function.

Figure 10.FigureThe microstructure10. The microstructure of the of the molded molded diffusion diffusion mixer mixer chip chip (550 (550°C, 0.5◦ C,MPa). 0.5 (a MPa).) Marco ( photo;a) Marco photo; (b) Enlarged area A; (c) Enlarged area B; (d) Profile A; (e) Enlarged area B. (b) Enlarged area A; (c) Enlarged area B; (d) Profile A; (e) Enlarged area B.

AFigure typical 10. Themicrostructure microstructure of of the the molded molded diffusionflow focusing mixer chipchip (550 is shown°C, 0.5 MPa). in Figure (a) Marco 11. Thephoto; macro A typicalphoto(b indicates microstructure) Enlarged that area the A; basic(c) ofEnlarged morphology the molded area B; (ofd) theProfile flow flow A; focusing focusing (e) Enlarged chip chip area was isB. achieved, shown as in shown Figure in Figure 11. The macro 11a. In order to provide more details about the formed microstructure, the area A and area B were photo indicates that the basic morphology of the flow focusing chip was achieved, as shown in enlarged,A typical as shown microstructure in Figure 11b,c,of the which molded are flow the combinationfocusing chip of is the shown three in channels Figure 11.at theThe bottom macro Figure 11a.andphoto In top, indicates order respectively. to that provide the They basic both more morphology demonstrate details of the aboutthat flow the theexpectedfocusing formed channels’chip was microstructure, achieved, morphology as shown was the generated, in area Figure A and area B were enlarged,which11a. In was order as deep shownto provideenough in formore Figure the details flow 11 focusing aboutb,c, which the experiment. formed are microstructure, the The combination extracted profilesthe area of are A the andshown three area in BFigure channels were at the bottom and11d,e,enlarged, top, and respectively. asthe shown section in profiles Figure They are11b,c, both both which characterized demonstrate are the combination by a that large the corner of expected the radius. three channels channels’ at the morphology bottom was and top, respectively. They both demonstrate that the expected channels’ morphology was generated, generated, which was deep enough for the flow focusing experiment. The extracted profiles are shown which was deep enough for the flow focusing experiment. The extracted profiles are shown in Figure in Figure 1111d,e,d,e, and and the the section section profiles profiles are both are characterized both characterized by a large corner by a radius. large corner radius.

Figure 11. The microstructure of the molded flow focusing chip (550 °C, 0.5 MPa). (a) Macro photo; (b) Enlarged area A; (c) Enlarged area B; (d) Profile A; (e) Profile B.

Figure 11.FigureThe microstructure11. The microstructure of the of the molded molded flow flow focusing focusing chip chip (550 (550 °C, 0.5◦C, MPa). 0.5 ( MPa).a) Macro ( aphoto;) Macro photo; (b) Enlarged area A; (c) Enlarged area B; (d) Profile A; (e) Profile B. (b) Enlarged area A; (c) Enlarged area B; (d) Profile A; (e) Profile B.

Micromachines 2018, 9, x 10 of 15

Micromachines 2018, 9, 269 10 of 15 A typical microstructure of the molded cell counting chip is shown in Figure 12. The macro photo indicates that the basic morphology of the cell counting chip was achieved, as shown in Figure 12a. The enlargedA typical microstructureimages of area of theA and molded area cell B countingare shown chip in is Figure shown 12b,c, in Figure respectively. 12. The macro The photo profile extractedindicates from that Figure the basic 12b morphologyis shown in ofFigure the cell 12d. counting Although chip wasthe line achieved, width as was shown only in 1.5 Figure μm 12 ona. the The enlarged images of area A and area B are shown in Figure 12b,c, respectively. The profile extracted mold, the molded groove is clear, with a depth of approximately 2 μm. Due to the contact between from Figure 12b is shown in Figure 12d. Although the line width was only 1.5 µm on the mold, the glass and the mold bottom, the top of the chip was relatively rough, which is beneficial for cells the molded groove is clear, with a depth of approximately 2 µm. Due to the contact between the glass to stayand inside. the mold According bottom, to the currently top of the morphology, chip was relatively the molded rough, whichglass is is potentially beneficial for valuable cells to stay for cell counting.inside. According to currently morphology, the molded glass is potentially valuable for cell counting.

◦ FigureFigure 12. The 12. microstructureThe microstructure of the of themolded molded cell cell counting counting chip chip (550 (550 °C,C, 0.5 0.5 MPa). MPa). (a (a)) Marco Marco photo; (b) (b) Enlarged area A; (c) Enlarged area B; (d) Profile A. Enlarged area A; (c) Enlarged area B; (d) Profile A.

3.3. Chip Application 3.3. Chip Application In order to verify the performance of the molded chips by the GMP, a mixed microfluidic chip Inwas order chosen to toverify conduct the theperformance filling microfluidic of the molded system experiment.chips by the In GMP, terms a of mixed the bonding microfluidic material, chip was chosenARseal PSAto conduct clear polypropylene the filling microfluidic film tapes (MH-90697, system experiment. Adhesives Research, In terms Inc., of Glen the bonding Rock, PA, material, USA) ARsealwere PSA used clear for theirpolypropylene simplicity, and film was tapes coated (MH with‐90697, an inert Adhesives silicone adhesive. Research, The Inc., tapes Glen provide Rock, an PA, USA)immediate were used bond for totheir most simplicity, materials and and can was also coated provide with a barrier an inert for preventing silicone adhesive. evaporation The and tapes providecross an contamination. immediate bond to most materials and can also provide a barrier for preventing evaporationThe and experiment cross contamination. of the filling microfluidic system is shown in Figure 13. Firstly, the corresponding Theliquid experiment inlet and the outletof the hole filling were fabricatedmicrofluidic by a lasersystem cutting is machiningshown in and Figure the bonding 13. Firstly, process the correspondingwas carried liquid out in inlet a clean and room, the outlet as shown hole in were Figure fabricated 13a. Next, by the a laser polyetheretherketone cutting machining (PEEK) and the connectors were bonded with the chip by glue, which helped to locate the inlet holes during the filling bonding process was carried out in a clean room, as shown in Figure 13a. Next, the with liquid, as shown in Figure 13b. Next, two inlet holes were filled with the red and blue ink by a polyetheretherketone (PEEK) connectors were bonded with the chip by glue, which helped to locate pump at the same filling speed of 2 µL/min. The filling status is shown in Figure 13c and the enlarged the inletimage holes of the during mixed the channel filling zone with is shownliquid, in as Figure shown 13 ind. Figure It was shown 13b. Next, that the two two inlet reagents holes can were flow filled with alongthe red the and channel blue withoutink by a cross pump contamination at the same occurring, filling speed and theof 2 obvious μL/min. stratification The filling phenomenon status is shown in Figurewas observed.13c and the This enlarged proves that image the moldedof the mixed chip can channel be used zone in a is microfluidic shown in Figure system. 13d. It was shown that the two reagents can flow along the channel without cross contamination occurring, and the obvious stratification phenomenon was observed. This proves that the molded chip can be used in a microfluidic system.

Micromachines 2018, 9, 269 11 of 15 Micromachines 2018, 9, x 11 of 15

(a) A schematic image (b) A physical image (c) Mix experiment (d) The enlarged image

Figure 13.13. TheThe experiment experiment of of filling filling the the mixing mixing microfluidic microfluidic system. system. (a) A schematic(a) A schematic image; (image;b) A physical (b) A physicalimage; (c )image; Mix experiment; (c) Mix experiment; (d) The enlarged (d) The image. enlarged image.

4. Discussions

4.1. Curved Side Wall Currently, only the curved sidewall of the microchannel could be achieved by the GMP in this experiment. Since Since the the cross cross-section‐section of of the the microstructure microstructure on on the the mold is rectangle, it is possible to obtain a side wall of the channels on the molded molded glass that is close close to to 90 90 when when appropriate appropriate parameters parameters are set. However,However, this this scenario scenario did did not not occur occur due due to to the the severe severe adhesion adhesion problem problem between between the moldthe mold and andthe glass the glass in the in actual the actual experiment. experiment. A promising A promising direction direction is the utilization is the utilization of the ultrasonic of the vibrationsultrasonic vibrationsduring the during GMP, which the GMP, is worth which exploring is worth in exploring the future. in the future. Although the curved side wall generated by the GMP is somewhat similar to the morphology obtained byby the the wet-etching, wet‐etching, the the GMP GMP has has obvious obvious advantages advantages over theover wet-etching the wet‐etching in several in aspects,several whichaspects, are which detailed are detailed below: below: (1) HigherHigher efficiency efficiency in in the the fabricating fabricating of of a singlea single microfluidic microfluidic glass glass chip chip The wet-etchingwet‐etching process process consists consists of cleaning,of cleaning, lithography, lithography, and bufferedand buffered oxide etchoxide (BOE) etch etching, (BOE) whichetching, needs which several needs instruments several instruments and is longer and thanis longer 1 h [43 than]. This 1 h process [43]. This can process achieve can the achieve production the productionof microfluidic of microfluidic glass chips atglass a commercial chips at a commercial scale on a regular scale on basis a regular and at basis reasonable and at reasonable costs. However, costs. However,for the fabricating for the fabricating of a single microfluidicof a single microfluidic glass chip, theglass time chip, of thethe GMPtime isof usuallythe GMP less is thanusually 15 min,less whichthan 15 is min, much which shorter is much than thatshorter of the than hydrofluoric that of the acidhydrofluoric (HF) etching. acid (HF) etching. (2) MoreMore eco-friendly eco‐friendly The chemical liquid used in the wet-etchingwet‐etching process, especially buffered buffered HF, HF, is harmful to environment, while only nitrogen is utilized in the GMP as a protecting gas, which is significantlysignificantly more eco-friendly.eco‐friendly. (3) WiderWider fabricating fabricating capacity capacity Some researchers [13–16] [13–16] have found that it is typically hard for the wet-etchingwet‐etching process to generate aa microstructuremicrostructure with with a depth-to-widtha depth‐to‐width ratio ratio that that is higher is higher than than 0.5, while0.5, while the GMP the hasGMP wider has widerfabricating fabricating capacity capacity than the than wet-etching. the wet‐etching. For instance, For instance, a microstructure a microstructure with a depth-to-width with a depth‐to ratio‐width of ratioone has of one been has achieved been achieved by the GMP by the [44 GMP]. [44]. In termsterms of of the the application application of channels of channels with awith curved a curved side wall, side many wall, researchers many researchers have conducted have conductedbiochemical biochemical tests on these. tests Lin on etthese. al. [ 43Lin] demonstrated et al. [43] demonstrated a micro flow-through a micro flow sampling‐through glass sampling chip, whichglass chip, was which fabricated was fabricated by wet-etching, by wet‐ andetching, the sideand the walls side of walls the channels of the channels were curved. were curved. The chipThe chipsampled sampled and separated and separated Cy5-labelled Cy5‐labelled BSA (Nanocs BSA (Nanocs Inc., New Inc., York, New NY, York, USA) andNY, anti-BSAUSA) and successfully. anti‐BSA successfully.Lee et al. [45] Lee demonstrated et al. [45] demonstrated a microcapillary a microcapillary electrophoresis electrophoresis (µ-CE) device for(μ‐ DNACE) device separation for DNA and separationdetection, andand detection, the channels and with the channels curved side with walls curved on side the walls plastic on chip the plastic were fabricated chip were byfabricated the hot byembossing the hot embossing method. Castaño- method.Á Castañolvarez et‐Álvarez al. [46] et fabricated al. [46] fabricated glass microfluidic glass microfluidic channels channels with curved with curved side walls by photolithography and wet‐etching. The microfluidic chips were successfully used, in combination with a metal‐wire end‐channel amperometric detector, for capillary

Micromachines 2018, 9, x 12 of 15 electrophoresisMicromachines 2018 (CE)., 9, 269 Evander et al. [47] fabricated glass microchannels with curved side12 walls of 15 by wet‐etching, and the glass microfluidic chip was used for acoustic force control of cells and particles in a sidecontinuous walls by microfluidic photolithography process. and Nevertheless, wet-etching. Thealthough microfluidic the side chips walls were of the successfully channel fabricated used, by thein combinationGMP were curved,with a metal-wire currently, end-channel the chips are amperometric still valuable detector, for many for capillaryapplications. electrophoresis (CE). Evander et al. [47] fabricated glass microchannels with curved side walls by wet-etching, and the 4.2. Bondingglass microfluidic Techniques chip was used for acoustic force control of cells and particles in a continuous microfluidic process. Nevertheless, although the side walls of the channel fabricated by the GMP were curved,Generally, currently, there the are chips three are categories still valuable of glass for many bonding applications. techniques: (1) Direct bonding, involving surface‐activated or fusion bonding; (2) Anodic bonding, with silicon as the intermediate layer; and (3) Adhesive4.2. Bonding bonding, Techniques with additional adhesive material. The strengths and weaknesses of each method Generally,can be found there in are [48]. three In categories this paper, of glass since bonding the focus techniques: is on (1)investigating Direct bonding, the involvingnew material performancesurface-activated in the orGMP, fusion the bonding; bonding (2) process Anodic bonding,was achieved with siliconby a assimple the intermediate method of layer;the glass microchanneland (3) Adhesive being bonding,sealed by with a polymer additional adhesive adhesive material.sheet. Compared The strengths with and the weaknesses three traditional of methods,each method the bonding can be process found in in this [48]. paper In this has paper, the following since the two focus advantages: is on investigating the new material performance in the GMP, the bonding process was achieved by a simple method of the (1) glassHigh microchannel efficiency being sealed by a polymer adhesive sheet. Compared with the three traditional methods,Compared the with bonding direct process and anodic in this paper bonding, has the the following polymer two adhesive advantages: sheet bonding can be achieved in room(1) High temperature efficiency without any additional chemical solution and, subsequently, there is no cloggingCompared problem, withwhich direct usually and anodic occurs bonding, in adhesive the polymer bonding. adhesive Thus, sheet it is bonding much faster can be to achieved achieve the prototypingin room temperature of the microfluidic without any chips additional for research. chemical solution and, subsequently, there is no clogging (2) problem,Recycling which value usually occurs in adhesive bonding. Thus, it is much faster to achieve the prototyping of the microfluidic chips for research. The bond of failed or used bonded microchips can easily be broken by soaking the microchips (2) Recycling value in acetone, with sonication, without damaging the glass microstructure, and they can be reused by the polymerThe bondadhesive of failed sheet or usedbonding bonded process. microchips can easily be broken by soaking the microchips in acetone,Although with it sonication,is a simple without method, damaging it is worth the glasspointing microstructure, out that the and bonding they can process be reused in bythis the paper polymer adhesive sheet bonding process. is assumed to not compromise the main advantages of the glass microfluidic chips over polymer Although it is a simple method, it is worth pointing out that the bonding process in this paper is chips, such as biocompatibility, optical transparency, and surface modification. As for assumed to not compromise the main advantages of the glass microfluidic chips over polymer chips, biocompatibility,such as biocompatibility, since the opticalanti‐reflection transparency, (AR) and film surface is designed modification. to be Asbiocompatible, for biocompatibility, the influence since of the ARthe anti-reflectionfilm is supposed (AR) film to be is designed small enough. to be biocompatible, In terms of the optical influence transparency, of the AR film it isis supposedtrue that the polymerto be smallAR film enough. may In exert terms ofsome optical negative transparency, influence it is true on thatthis the aspect. polymer However, AR film may this exert issue some can be resolvednegative if the influence test is on conducted this aspect. by However, placing this the issue chip can upside be resolved down, if as the shown test is conducted in Figure by 14. placing When the surfacethe chipmodification upside down, is concerned, as shown in since Figure there 14. When are still the surfacethree glass modification surfaces is concerned,left in the sincemicrochannel, there differentare still surface three glass modification surfaces left procedures in the microchannel, can be conducted different surface as well, modification such as protein procedures immobilization can be [49].conducted Therefore, as the well, microfluidic such as protein chip immobilization assembled by [49 polymer]. Therefore, adhesive the microfluidic sheet bonding chip assembled is still valuable by for practicalpolymer adhesiveapplications. sheet bonding is still valuable for practical applications.

FigureFigure 14. 14.A typicalA typical testing testing diagram diagram of of the the produced glass glass and and anti-reflection anti‐reflection (AR) (AR) film film chip. chip.

5. Conclusions This paper investigated the fabricating of glass microfluidic chips by the GMP, which provided a promising method for fast prototyping and mass production of microfluidic chips in the future. The main conclusions were as follows:

Micromachines 2018, 9, 269 13 of 15

5. Conclusions This paper investigated the fabricating of glass microfluidic chips by the GMP, which provided a promising method for fast prototyping and mass production of microfluidic chips in the future. The main conclusions were as follows:

(1) A groove with a 60 µm height could be obtained by the GMP in 12 min, and the bottom roughness, Ra, could be as small as 10 nm. The molded groove depth increased dramatically with the molding temperature and pressure before the time when the glass contacted with the bottom of the mold. Precautions should be taken to avoid cracks generated inside the glass. (2) The microstructure of a diffusion mixer chip and a flow focusing chip were generated with more than 40 µm groove depths by the GMP, and the microstructure of the cell counting chip was fabricated with the groove of a 2 µm depth and 1.5 µm width. (3) The study of mold wear indicated that the macro shape of the microstructure on the mold changed little over the time of 20 experiments, although the edge became a little blunt due to the weak stiffness in this area. The results from the energy-dispersive X-ray spectroscopy (EDS) analysis found that there was no chemical wear. (4) The performance verification of the molded chips was conducted on a mixed microfluidic chip and the result indicated that the two reagents could flow along the channel without cross contamination, and that the obvious stratification phenomenon was observed. This proved that the molded chip could be used in a microfluidic system.

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-666X/9/6/269/s1, Figure S1: The morphology of the mold for fabricating channels on glass, Figure S2: The morphology of three different molds for fabricating microfluidic chips, Figure S3: The mold morphology after GMP, Figure S4: The microstructure of molds before GMP, Figure S5: The microstructure of the molds after GMP. Author Contributions: J.C. conceived and designed the experiments; T.W. performed the experiments and wrote the paper; T.Z. provided theoretical guidance; L.S. contributed to the fabrication of the core mold. Acknowledgments: This project is supported by the National Natural Science Foundation of China (Grant No. U1537208), the National High Technology Research and Development Program of China (863) Grant No. 2015AA043601 and the National Natural Science Foundation of China (Grant No. 51375050). Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

References

1. Kkr, T.; Vijayalakshmi, M.A. A review on recent developments for biomolecule separation at analytical scale using microfluidic devices. Anal. Chem. Acta 2016, 906, 7–21. 2. Geigy, C. Capillary electrophoresis and sample injection systems integrated on a planar glass chip. Anal. Chem. 1992, 64, 1928–1932. 3. Seiler, K.; Harrison, D.J.; Manz, A. Planar glass chips for capillary electrophoresis: Repetitive sample injection, quantitation, and separation efficiency. Anal. Chem. 1993, 65, 1481–1488. [CrossRef] 4. Kricka, L.J.; Faro, I.; Heyner, S.; Garside, W.T.; Fitzpatrick, G.; McKinnon, G.; Ho, J.; Wilding, P. Micromachined analytical device: Microchips for semen testing. J. Pharm. Biomed. 1997, 15, 1443–1447. [CrossRef] 5. Jacobson, S.C.; Hergenröder, R.; Koutny, L.B.; Ramsey, J.M. Open channel electrochromatography on a microchip. Anal. Chem. 1994, 66, 2369–2373. [CrossRef] 6. Effenhauser, C.S.; Paulus, A.; Manz, A.; Widmer, H.M. High-speed separation of antisense oligonucleotides on a micromachined capillary electrophoresis device. Anal. Chem. 1994, 66, 2949–2953. [CrossRef] 7. Shoffner, M.A.; Cheng, J.; Hvichia, G.E.; Kricka, L.J.; Wilding, P. Chip PCR. I. Surface passivation of microfabricated silicon–glass chips for PCR. Nucleic Acids Res. 1996, 24, 375–379. [CrossRef][PubMed] 8. Yu, L.; Huang, H.; Dong, X.; Wu, D.; Qin, J.; Lin, B. Simple, fast and high throughput single cell analysis on PDMS microfluidic chips. Electrophoresis 2008, 29, 5055–5060. [CrossRef][PubMed] Micromachines 2018, 9, 269 14 of 15

9. Hong, T.F.; Ju, W.J.; Wu, M.C.; Tai, C.H.; Tsai, C.H.; Fu, L.M. Rapid prototyping of PMMA microfluidic chips utilizing a CO2 laser. Microfluid. Nanofluid. 2010, 9, 1125–1133. [CrossRef] 10. Fu, L.M.; Ju, W.J.; Yang, R.J.; Wang, Y.N. Rapid prototyping of glass-based microfluidic chips utilizing

two-pass defocused CO2 laser beam method. Microfluid. Nanofluid. 2013, 14, 479–487. [CrossRef] 11. Rodriguez, I.; Spicar-Mihalic, P.; Kuyper, C.L.; Fiorini, G.S.; Chiu, D.T. Rapid prototyping of glass microchannels. Anal. Chim. Acta 2003, 496, 205–215. [CrossRef] 12. Chen, Q.; Li, G.; Jin, Q.H.; Zhao, J.L.; Ren, Q.S.; Xu, Y.S. A rapid and low-cost procedure for fabrication of glass microfluidic devices. J. Microelectromech. Syst. 2007, 16, 1193–1200. [CrossRef] 13. Grétillat, M.A.; Paoletti, F.; Thiébaud, P.; Roth, S.; Koudelka-Hep, M.; De Rooij, N.F. A new fabrication method for capillary tubes with lateral insets and outlets. Sens. Actuators A Phys. 1997, 60, 219–222. [CrossRef] 14. Bu, M.; Melvin, T.; Ensell, G.J.; Wilkinson, J.S.; Evans, A.G.R. A new masking technology for deep glass etching and its microfluidic application. Sens. Actuators A Phys. 2004, 115, 476–482. [CrossRef] 15. Mourzina, Y.; Steffen, A.; Offenhäusser, A. The evaporated metal masks for chemical glass etching for BioMEMS. Microsyst. Technol. 2005, 11, 135–140. [CrossRef] 16. Zhu, H.; Holl, M.; Ray, T.; Bhushan, S.; Meldrum, D.R. Characterization of deep wet etching of fused silica glass for single cell and optical sensor deposition. J. Micromech. Microeng. 2009, 19, 65013–65020. [CrossRef] 17. Ronggui, S.; Righini, G.C. Characterization of reactive ion etching of glass and its applications in integrated optics. J. Vac. Sci. Technol. 1991, 9, 2709–2712. [CrossRef] 18. Zeze, D.A.; Forrest, R.D.; Carey, J.D.; Cox, D.C.; Robertson, I.D.; Weiss, B.L.; Silva, S.R.P. Reactive ion etching of quartz and for microelectronic applications. J. Appl. Phys. 2002, 92, 3624–3629. [CrossRef] 19. Ichiki, T.; Sugiyama, Y.; Ujiie, T.; Horiike, Y. Deep dry etching of borosilicate glass using fluorine-based high-density plasmas for microelectromechanical system fabrication. J. Vac. Sci. Technol. 2003, 21, 2188–2192. [CrossRef]

20. Park, J.H.; Lee, N.E.; Lee, J.; Park, J.S.; Park, H.D. Deep dry etching of borosilicate glass using SF6 and SF6/Ar inductively coupled plasmas. Microelectron. Eng. 2005, 82, 119–128. [CrossRef]

21. Leech, P.W. Reactive ion etching of quartz and silica-based in CF4/CHF3 plasmas. Vacuum 1999, 55, 191–196. [CrossRef] 22. Yen, M.H.; Cheng, J.Y.; Wei, C.W.; Chuang, Y.C.; Young, T.H. Rapid cell-patterning and microfluidic chip

fabrication by crack-free CO2 laser ablation on glass. J. Micromech. Microeng. 2006, 16, 1143–1153. [CrossRef] 23. Sohn, I.B.; Lee, M.S.; Woo, J.S.; Lee, S.M.; Chung, J.Y. Fabrication of photonic devices directly written within glass using a femtosecond laser. Opt. Express 2005, 13, 4224–4229. [CrossRef][PubMed] 24. Marcinkeviˇcius,A.; Juodkazis, S.; Watanabe, M.; Miwa, M.; Matsuo, S.; Misawa, H.; Nishii, J. Femtosecond laser-assisted three-dimensional microfabrication in silica. Opt. Lett. 2001, 26, 277–279. [CrossRef] 25. Nikumb, S.; Chen, Q.; Li, C.; Reshef, H.; Zheng, H.Y.; Qiu, H.; Low, D. Precision glass machining, drilling and profile cutting by short pulse laser. Thin Solid Films 2005, 477, 216–221. [CrossRef] 26. Arif, M.; Rahman, M.; San, W.Y.; Doshi, N. An experimental approach to study the capability of end-milling for microcutting of glass. Int. J. Adv. Manuf. Technol. 2011, 53, 1063–1073. [CrossRef] 27. Arif, M.; Rahman, M.; San, W.Y. Ultraprecision ductile mode machining of glass by micromilling process. J. Manuf. Process. 2011, 13, 50–59. [CrossRef] 28. Slikkerveer, P.J.; Bouten, P.C.P.; De Haas, F.C.M. High quality mechanical etching of brittle materials by powder blasting. Sens. Actuators A Phys. 2000, 85, 296–303. [CrossRef] 29. Schlautmann, S.; Wensink, H.; Schasfoort, R.; Elwenspoek, M.; Van Der Berg, A. Powder-blasting technology as an alternative tool for microfabrication of capillary electrophoresis chips with integrated conductivity sensors. J. Micromech. Microeng. 2001, 11, 386. [CrossRef] 30. Plaza, J.A.; Lopez, M.J.; Moreno, A.; Duch, M.; Cane, C. Definition of high aspect ratio columns. Sens. Actuators A-Phys. 2003, 105, 305–310. [CrossRef] 31. Yan, B.H.; Wang, A.C.; Huang, C.Y.; Huang, F.Y. Study of precision micro-holes in borosilicate glass using micro EDM combined with micro ultrasonic vibration machining. Int. J. Mach. Tools Manuf. 2002, 42, 1105–1112. [CrossRef] 32. Dietrich, T.R.; Ehrfeld, W.; Lacher, M.; Krämer, M.; Speit, B. Fabrication technologies for microsystems utilizing photoetchable glass. Microelectron. Eng. 1996, 30, 497–504. [CrossRef] Micromachines 2018, 9, 269 15 of 15

33. Yan, J.; Zhou, T.; Yoshihara, N.; Kuriyagawa, T. Shape transferability and microscopic deformation of molding dies in aspherical glass lens molding press. J. Manuf. Technol. Res. 2009, 1, 85–102. 34. Zhou, T.; Yan, J.; Masuda, J.; Oowada, T.; Kuriyagawa, T. Investigation on shape transferability in ultraprecision glass molding press for microgrooves. Precis. Eng. 2011, 35, 214–220. [CrossRef] 35. Kobayashi, R.; Zhou, T.; Shimada, K.; Mizutani, M.; Kuriyagawa, T. Ultraprecision glass molding press for microgrooves with different pitch sizes. Int. J. Automot. Technol. 2013, 7, 678–685. [CrossRef] 36. Yi, A.Y.; Chen, Y.; Klocke, F.; Pongs, G.; Demmer, A.; Grewell, D.; Benatar, A. A high volume precision compression molding process of glass diffractive optics by use of a micromachined fused silica wafer mold and low Tg optical glass. J. Micromech. Microeng. 2006, 16, 2000–2005. [CrossRef] 37. Huang, C.Y.; Hsiao, W.T.; Huang, K.C.; Chang, K.S.; Chou, H.Y.; Chou, C.P. Fabrication of a double-sided micro-lens array by a glass molding technique. J. Micromech. Microeng. 2011, 21, 1–6. [CrossRef] 38. Saotome, Y.; Imai, K.; Sawanobori, N. Microformability of optical glasses for precision molding. J. Mater. Process. Technol. 2003, 140, 379–384. [CrossRef] 39. Chen, Q.; Chen, Q.; Maccioni, G. Fabrication of microfluidics structures on different glasses by simplified imprinting technique. Curr. Appl. Phys. 2013, 13, 256–261. [CrossRef] 40. Chen, Q.; Chen, Q.; Maccioni, G.; Sacco, A.; Ferrero, S.; Scaltrito, L. Fabrication of microstructures on glass by imprinting in conventional furnace for lab-on-chip application. Microelectron. Eng. 2012, 95, 90–101. [CrossRef] 41. Huang, C.Y.; Kuo, C.H.; Hsiao, W.T.; Huang, K.C.; Tseng, S.F.; Chou, C.P. Glass biochip fabrication by laser micromachining and glass-molding process. J. Mater. Process. Technol. 2012, 212, 633–639. [CrossRef] 42. Zhu, X.Y.; Wei, J.J.; Chen, L.X.; Liu, J.L.; Hei, L.F.; Li, C.M.; Zhang, Y. Anti-sticking Re-Ir coating for glass molding process. Thin Solid Films 2015, 584, 305–309. [CrossRef] 43. Lin, C.H.; Lee, G.B.; Lin, Y.H.; Chang, G.L. A fast prototyping process for fabrication of microfluidic systems on soda-lime glass. J. Micromech. Miceoeng. 2001, 11, 726–732. [CrossRef] 44. Takahashi, M.; Sugimoto, K.; Maeda, R. Nanoimprint of glass materials with glassy carbon molds fabricated by focused-ion-beam etching. Jpn. J. Appl. Phys. 2005, 44, 5600–5605. [CrossRef] 45. Lee, G.B.; Chen, S.H.; Huang, G.R.; Sung, W.C.; Lin, Y.H. Microfabricated plastic chips by hot embossing methods and their applications for dna separation and detection. Sens. Actuators B Chem. 2001, 75, 142–148. [CrossRef] 46. Castaño-Álvarez, M.; Ayuso, D.F.P.; Granda, M.G.; Fernández-Abedul, M.T.; García, J.R.; Costa-García, A. Critical points in the fabrication of microfluidic devices on glass substrates. Sens. Actuators B Chem. 2008, 130, 436–448. [CrossRef] 47. Evander, M.; Lenshof, A.; Laurell, T.; Nilsson, J. Acoustophoresis in wet-etched glass chips. J. Anal. Chem. 2008, 80, 5178–5185. [CrossRef][PubMed] 48. Iliescu, C.; Taylor, H.; Avram, M.; Miao, J.; Franssila, S. A practical guide for the fabrication of microfluidic devices using glass and silicon. Biomicrofluidics 2012, 6, 016505. [CrossRef][PubMed] 49. Qin, M.; Hou, S.; Wang, L.; Feng, X.; Wang, R.; Yang, Y.; Wang, C.; Yu, L.; Shao, B.; Qiao, M. Two methods for glass surface modification and their application in protein immobilization. Colloids Surf. B Biointerfaces 2007, 60, 243–249. [CrossRef][PubMed]

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