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Multi-layer hierarchical array fabricated with diatom frustules for highly sensitive bio-detection

applications

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2014 J. Micromech. Microeng. 24 025014

(http://iopscience.iop.org/0960-1317/24/2/025014)

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Journal of Micromechanics and Microengineering

J. Micromech. Microeng. 24 (2014) 025014 (7pp) doi:10.1088/0960-1317/24/2/025014

Multi-layer hierarchical array fabricated withdiatom frustules for highly sensitivebio-detection applicationsAobo Li, Jun Cai, Junfeng Pan, Yu Wang, Yue Yue and Deyuan Zhang

Bionic and Micro/Nano/Bio Manufacturing Technology Research Center, School of MechanicalEngineering and Automation, Beihang University, No. 37 Xueyuan Road, HaiDian District,Beijing 100191, Peoples Republic of ChinaE-mail: jun_cai@buaa.edu.cn

Received 4 September 2013, revised 17 November 2013Accepted for publication 22 November 2013Published 15 January 2014

AbstractDiatoms have delicate porous structures which are very beneficial in improving the absorbingability in the bio-detection field. In this study, multi-layered hierarchical arrays were fabricatedby packing Nitzschia soratensis (N. soratensis) frustules into Cosinodiscus argus (C. argus)frustules to achieve advanced sensitivity in bio-detection chips. Photolithographic patterningwas used to obtain N. soratensis frustule arrays, and the floating behavior of C. argus frustuleswas employed to control their postures for packing N. soratensis frustule array spots. Themorphology of the multi-layer C. argusN. soratensis package array was investigated byscanning electron microscopy, demonstrating that the overall and sub-structures of the diatomfrustules were retained. The signal enhancing effect of multi-layer C. argusN. soratensispackages was demonstrated by fluorescent antibody test results. The mechanism of theenhancement was also analyzed, indicating that both complex hierarchical frustule structuresand optimized posture of C. argus frustules were important for improving bio-detectionsensitivities. The technique for fabricating multi-layer diatom frustules arrays is also useful formaking multi-functional biochips and controllable drug delivery systems.

Keywords: diatom frustules, multi-layer, hierarchical, bio-detection, sensing

S Online supplementary data available from stacks.iop.org/JMM/24/025014/mmedia

(Some figures may appear in colour only in the online journal)

1. Introduction

Diatoms are microscopic, single-celled photosynthetic algaewidely distributed in both freshwater and seawater, formingabout 100 000 different species ranging from 1 m to 4 mm insize with thousands of different morphologies [1, 2]. Diatomshave distinct 3D architectures of their silica microshells (alsoknown as frustules) with highly ordered pore structures. Thesehierarchical pore structures have unique mechanical, photonic,optical and molecular transporting properties [36]. Evenwith the highly developed microscale/nanoscale fabricationtechnologies nowadays, it is still difficult to imitate intricate

structures and morphologies like naturally developed diatomfrustules.

Being attracted and inspired by the exquisite structureof diatom frustules, researchers started a new research areanow called diatom nanotechnology about 25 years ago [7]. Aseries of research studies on diatom frustules have been carriedout and many promising potential applications have beendemonstrated [8], including (yet not limited to) filtration [6, 9],sensing [6, 1013], electrochemical/optical biomoleculediagnostic devices [1416], optics/photovoltaic devices [17]and drug delivery systems [18, 19]. However, most of theseefforts focused on mass randomly distributed diatoms or singlediatoms [19], and there were few reports on manipulating or

0960-1317/14/025014+07$33.00 1 2014 IOP Publishing Ltd Printed in the UK

J. Micromech. Microeng. 24 (2014) 025014 A Li et al

Figure 1. Schematic illustrations of the fabrication process of the C. argusN. soratensis package array.

arranging diatoms to achieve well-arranged diatom frustules,except by hand as an art form [20].

Frustules have been frequently used as signal enhancingdevices in biosensors [1316]. For high-throughput biosensorsbased on diatoms, precise manipulation and arrangement ofdiatom frustules on chips can be critical to improving theaccuracy and efficiency of a test. In our previous studies, wehave successfully fabricated microscale Cymbella perpusilla,Cosinodiscus argus (C. argus) and Nitzschia dubia frustulearrays on glass substrates using photolithography technology.These studies have shown the existence of an enhancedabsorbing effect of frustules on target molecules [21]. In thispaper, with a novel method, we further explored and analyzedthe enhancing effects. Chips combining differently sizeddiatom frustules were made, on which multi-layered diatomfrustule package arrays were fabricated to further improve thesensitivity of the chips for bio-detection applications.

2. Materials and methods

2.1. Diatom frustules preparation

Diatoms used in the study were C. argus, provided byXiamen University and Nitzschia soratensis (N. soratensis,provided by the Freshwater Algae Culture Collection ofthe Institute of Hydrobiology (the FACHB-collection) ofChinese Academy of Sciences). C. argus diatoms werecultured in D1 medium (the formula was from FACHB-collection, attached in supplementary materials (available atstacks.iop.org/JMM/24/025014/mmedia)) and N. soratensisdiatoms were cultured in f/2 medium [22] at 20 C. Thediatoms were cultured in a light incubator (natural light, 16 000lm, incubator type MLR-352, Panasonic Healthcare Co., Ltd,Japan) for 12:12 h light/dark light periods. 25 ml diatomstrains were added into another 25 ml culture media in a250 ml conical flask. The culture media volume was doubledevery week to keep diatom reproduction rate high, and thecontainer was also changed from 250 to 2000 ml accordingly.Six weeks later, the whole culture media reached 1600 ml,and the density of diatoms reached a relatively high level (the

density of C. argus was around 4000 cells ml1 and the densityof N. soratensis was about 5.5 105 cells ml1).

One liter of C. argus diatoms culture media and 400 mlof N. soratensis diatoms culture media were concentratedthrough repeated settling and separation, and then treated withsulfuric acid. Four hundred milliliters of concentrated sulfuricacid was used in cleaning C. argus diatoms. After the acidwas added into the concentrated diatom medium, the solutionwas heated in a water bath at 65 C for 40 min. Frustuleswere then separated from the solution by repeated dilutingand settling processes [23]. For N. soratensis diatoms, 450 mlconcentrated sulfuric acid was added. The solution was heatedat 65 C for 10 min. N. soratensis frustules were separatedfrom the solution by repeated diluting and centrifuging(4000 rpm). The concentrated solutions of cleaned frustulesof C. argus and N. soratensis were dropped onto clean glassslides respectively. After the water evaporated, the specimenwas coated with a thin gold film (1020 nm) and inspectedwith SEM (scanning electron microscopy) (CS-3400, ObducatCamScan Ltd, Cambridge, CAMBS, UK) to investigate theirmorphologies.

2.2. Fabrication of the C. argusN. soratensis frustulepackage array

The process of fabricating the C. argusN. soratensis frustulepackage array is shown in figure 1. First, N. soratensisfrustule arrays were prepared by using the template assistantmethod [21]. 26 mm 26 mm glass slides were coatedwith hot-melt adhesive ethylene-vinyl acetate copolymer(EVA, type A-1, obtained from Sidelong Corporation, Wuhan,China), then a positive photoresist (BP21237S, KempurMicroelectronics. Inc., Beijing, China) coating was addedwith a spin coater, the thickness of which was adjustedto 23 m. Later, the photolithography was carried outfor 20 s (lithography machine type RMA2000, picturesof the mask can be seen in supplementary materials(available at stacks.iop.org/JMM/24/025014/mmedia)). Afterbeing flushed with the photoresist developer, the patterned pitsarray was achieved. The diameter of the pits was designedto be 60 m. Then, 0.5 ml condensed suspension of N.

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Figure 2. Different types of array for fluorescent antibody detection.

soratensis frustules was dropped onto the substrate, coveringthe pits and its surrounding area. After being dried, thesubstrates were reheated to 140 C for 2 min to bond thefrustules to the substrate. The diatoms that scattered aroundthe pits and not bonded to the EVA substrate were blown awaywith compressed N2 flow. Finally, the array of N. soratensisfrustules was achieved.

Secondly, the secondary photolithography was carriedout for the packing process. A similar patternedmask (figure 1 in supplementary materials (available atstacks.iop.org/JMM/24/025014/mmedia)) was used to makehomocentric pits around the frustule spots, yet the diameterof the pits was amplified to 200 m. After the exposure andetching, EVA around the N. soratensis spots was exposed,leaving enough space for the C. argus assembly. C. argusfrustules were manipulated and uniformly posed for packing.The frustule posture control was based on floating the dryclean frustules on a water surface [24]. Twenty microlitersof deionized water was dropped on a flat PDMS surface,which formed a round droplet since the PDMS is hydrophobic.Hundreds of C. argus frustules were attached onto the surfaceof a glass needle first, and then the diatoms were dispersedonto the droplet surface by tapping the glass needle above thedroplet. About half of the frustules settled to the bottom of thedroplet, and another half floated on the surface of the dropletwith their concave side up, like boats on the water. Thesefloating frustules tended to assemble and form a compact filmon the droplet surface.

Then the as-prepared chip with the array of N. soratensisfrustules was inverted and made to touch the droplet coveredwith C. argus frustules. After the array area contacted thedroplet surface, the chip was gently removed. Then most ofthe floating frustules were transferred and attached to the arrayarea of the chip. After being dried and slightly adjusted by amicromanipulator, the chip was heated at 140 C for 2 min tomake the C. argus frustules in the pits bond to the EVA layerof the substrate. The chip was blown with compressed N2 flowagain to remove loose C. argus frustules, only the frustulesbonded in the pits were left, containing N. soratensis frustulesinside.

2.3. Fluorescent antibody detection

The solutions for antibody bonding were preparedin a sterile environment. 50 ml 2% solution of 3-aminopropyltriethoxysilane (APS) in acetone and 100 mlsolution of purified primary rabbit immunoglobulin G (IgG)(obtained from Life Technology Corporation, catalog no. 026102, diluted to 5 g ml1 in phosphate buffered saline) wereprepared. 25 ml solution of fluorescent secondary donkey anti-rabbit IgG (obtained from Life Technologies Corporation,catalog no. A21206, labeled by Alexa Fluor R 488,

approximate fluorescence excitation maxima 495 nm andemission maxima 519 nm, diluted to 10 g ml1 in phosphatebuffered saline) was prepared in a dark room [14].

The substrates were immersed into the APS solution for90 s for surface modification, and then they were washedwith acetone for 1 min. After being dried, the substrates wereimmersed into the rabbit IgG solution at 38 C for 12 h forincubation. Later, the substrates were washed with deionizedwater for 1 min and then immersed into the donkey anti-rabbitIgG solution in a dark room for 2 h, during which donkey anti-rabbit IgG would bind to rabbit IgG. In the end, the substrateswere washed with deionized water for 1 min and then observedwith a fluorescent microscope (XSY-1, Chongqing Optical &Electrical Instrument Co., Ltd, Chongqing, China).

To evaluate the enhancement of the fluorescent signal forthe multi-layered C. argusN. soratensis package array, someother chips were also prepared for comparison. Figure 2 showsthe schematic diagram of different array types; one blank platewith EVA coating was also prepared as the control sample. Thesample preparation process was the same as the multi-layeredsample.

3. Results and discussions

3.1. Morphology of diatom frustules

The morphology of C. argus frustules was observed with SEMas shown in figure 3. The overall shape of C. argus frustuleswas bowl-like, and the average diameter was around 80100 m. In figures 3(a) and (d), the frustules appeared ellipticalbecause the figures were the side views of them. Figures 3(a)(c) show the morphology of a C. argus frustule with its convexside up, from which we can see that the frustule surface wassmooth. Sub-micron elliptical holes (major diameter around300 nm, minor diameter around 170 nm) and nanopores(known as cribellum, about 90100 nm in diameter) wereuniformly distributed over it. Figures 3(d)( f ) display themorphology of a C. argus frustule with its concave side up. Itsbowl-like structure was more clearly demonstrated, and therewere small spines around the inside edge. In the enlargedview (figures 3(e) and ( f )), the uniform microholes (knownas areola pores, about 0.40.8 m in diameter) were radiallydistributed about the center of the valve, with nano cribellumpores at the bottom of them. Figure 3 illustrates that theC. argus frustules themselves were micronano hierarchicallystructured. The height of the frusutle was about 1522 m andthe thickness of the frusutle wall was about 1.52.2 m. Thisbowl-like structure makes it possible to use C. argus frustulesas containers to pack other microparticles inside if they canbe bonded onto the substrate with their concave side facingtoward the substrate.

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(a)

(d )

(b)

(e) (f )

(c)

Figure 3. SEM images of C. argus diatom frustules: (a) a convex side up frustule; (b) the external porous layer of cribellum; (c) cribellumpores are clean; (d) a concave side up frustule; (e) the internal porous layer of areola; ( f ) areola pores were intact and clean.

(a) (b)

Figure 4. SEM images of N. soratensis diatom frustules.

The morphology of N. soratensis frustules is shownin figure 4. These frustules resemble tiny kidney beans(figure 4(b)), with the long axis about 1015 m and shortaxis around 5m. There were also nanopores (60 nm80 nmin diameter) on their surface, giving them huge surface area.Most of them tended to split into two valves after the acidcleaning process, only a very few still kept as a whole.

3.2. Package array of C. argusN. soratensis

Figure 5 displays different stages of the array fabrication.Figure 5(a) shows the array of assembled N. soratensis frustulespots ready for packing which were well organized. Figure 5(d)shows the detailed morphology of one spot of assembledN. soratensis. In figure 5(d), N. soratensis were randomlydistributed bonded to the substrate. Figure 5(b) shows theresult of packing N. soratensis with C. argus. All C. argusfrustules concave sides were facing toward the substrate. Inaddition, since N. soratensis frustule spot diameter was 60 mwhile the average diameter of C. argus was around 100 m, N.soratensis frustules in each spot were fully covered by C. argusfrustules. Figure 5(c) demonstrates the detailed morphologyof the outer surface of one package spot. Figure 5(e) showsthe overview of one C. argusN. soratensis package spot. Infigures 5(c) and (e), the whole structure of C. argus was intact

and the surface was kept clean, indicating that the packingprocedure did not damage the frustule structures. During thepacking process, about half of the C. argus frustules did notlocate exactly on the top of the arrayed N. soratensis frustules,so micromanipulation was used to adjust their positions beforebonding. One C. argus frustule was broken and pushed awaywith a micro glass needle mounted on the micromanipulator(figure 5( f )) to further illustrate the multi-layer structure of thepackage of C. argusN. soratensis. Covered by C. argus, N.soratensis frustules were well kept in their initial morphologyand remained functional. The remaining confined fragmentring of the C. argus frustule was kept as a whole, indicatingthat the edge of the C. argus frustule was firmly bonded to thesubstrate with no gap.

3.3. Fluorescent antibody detection results

The results of fluorescent antibody detections on the multilayerpackage and corresponding control group are shown infigure 6. After the surfaces of substrates were modified byAPS, amino groups were attached to frustule surfaces throughalkoxy groups. After the modified substrates were immersedinto the rabbit IgG solution, the aldehydes and carboxylgroups from IgG s carbohydrate will react with the aminogroups, allowing sufficient rabbit IgG to bond to the modified

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(d ) (e) (f )

(a) (b) (c)

Figure 5. Packaging of N. soratensis frustules: (a) array of N. soratensis frustules; (b) array of C. argusN. soratensis packages; (c) detailedmorphology of the covering C. argus; (d) a single spot of N. soratensis frustules array; (e) a single spot of package array; ( f ) N. soratensisfrustules exposed after the covering C. argus frustule was broken and pushed away.

(a) (b) (c) (d ) (e)

Figure 6. Typical array spots after fluorescent antibody detection: (a) C. argusN. soratensis package; (b) N. soratensis frustules; (c) C.argus frustules (concave-down); (d) C. argus frustules (convex-down); (e) blank (EVA surface).

surface. Then molecules on substrates reacted with sufficientfluorophore-labeled donkey anti-rabbit IgG. In fluorescentfigures (figure 6), the labeled IgG showed the position and theamount of bonded rabbit IgG. Typically, if a detection spot hasmore functional nanopores, then it could bond more proteinsbecause of its high area surface, leading to a fluorescenceenhancement in bio-detections [14].

Fluorescent images were further analyzed with Image Pro-Plus 6.0 to obtain the mean fluorescence density (MFD) ofarray spots. For each substrate, the MFD of the array spot wascalculated by dividing integrated optical density (IOD) withthe fluorescence area of the array spot:

MFD = IODFluorescence area

. (1)Here, both the IOD and fluorescence area of the array

spot are restricted to the marked fluorescence area only, theborder line of which is shown in the main view of figure 6(redoutlines), so as to keep the comparison meaningful and fair.The MFD of a substrate is a direct parameter to judge its abilityto bond target molecules. The statistics of MFD are shown intable 1 (with standard deviation, 36 sets of data from eachgroup were extracted).

It can be concluded from table 1 that the C. argusN.soratensis packages generated the brightest fluorescence; thisis due to the coupling enhancement effect of C. argusN.

soratensis packages, which integrated the surface areas of twokinds of diatom frustules in an optimized way. In addition, italso indicated that the C. argus frustules did not filter targetmolecules with their hierarchical holes, allowing frustulesinside to make contact with IgG molecules. The signal of bothconcave-down and convex-down C. argus was a little higherthan that of N. soratensis. This might be caused by the morecomplex and hierarchical structures of C. argus frustules. Theirposture also affected the detection result. The mean opticaldensity of concave-down frustules was higher than that of theconvex-down frustules, indicating that the nanopores of theirouter frustule surface played a more important role in bondingtarget molecules. When C. argus frustule were concave-down,all nanopores were exposed to the target molecule; yet forconvex-down frustules, some nanopores were sealed whenthey were bonded to substrates (figure 3). These results notonly further proved the validity of the enhancing mechanism ofnanopores on the target molecules bonding, but also indicatedthat the complex hierarchical structures and optimized posturewere both important for enhancing signals in a bio-detection,thus improving the detection sensitivities.

3.4. Potential and future plan

Based on the techniques discussed above, some advancedprocessing steps can be further developed to fabricate more

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Figure 7. Schematic of potential use of the packaging method.

Table 1. Results of fluorescent antibody tests.

Each groups MDF ( 102)C. Argus Nitzschia Sp. Nitzschia Sp. C. Argus frustules C. Argus frustules Blank

Group package frustules (concave-down) (convex-down) (EVA surface)Average 13.02 0.89 4.82 0.39 8.26 0.68 5.00 0.48 0.94 0.09

sophisticated biosensors. For example, if we use Cosinodiscussp. [9], which has a similar shape and structure with C. Argusand smaller nano cribellum pores on the outer surface toreplace C. Argus, and modify the surface of N. soratensisfrustules and Cosinodiscus sp. frustules separately [25], thepackages will achieve integrated functions: both filtering anddetecting (figure 7). With such devices, only the moleculesthrough the pores of the Cosinodiscus sp. frustules can bedetected, which will improve the accuracy and efficiency of thetest. This is similar to figure 2 in [2], but with the improvementthat the biosensors are themselves diatoms, and thus havea large surface area. With pore enlargement/narrowingtechniques [26, 27], the pore of frustules can be adjustedfor detecting targets of different sizes. Also, the frustulesof Cosinodiscus sp. and N. soratensis can be modified withvarious probes to detect different targets. The technologyof fabricating multi-layer hierarchical arrays with diatomfrustules may also be further adjusted and improved for targetdrug delivery systems, by packing nano drug particles orbiomacromolecules inside the outer shell for sustained releaseapplications.

4. Conclusions

In this study, we propose a method to achieve advancedsensitivity in bio-detection chips by building multi-layer meso-structured arrays using C. argus and N. soratensis frustules.With a double photolithography process and a posture controlmethod based on C. argus frustules floating on the surface of asessile water droplet, frustules of N. soratensis were packagedinto C. argus frustules. The fluorescent antibody test resultsshowed that the fluorescent intensities of these multi-layer C.argusN. soratensis packages were at least 2.7 times strongerthan those samples with only one type of diatom frustules.

The mechanism was also analyzed, which showed that thecomplex hierarchical structure and posture are both importantfor the detection sensitivity. Such processing techniques canbe used for fabricating highly sensitive testing arrays and novelfunctional devices.

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (No. 51322503, 51275025, 51245014)and Program for New Century Excellent Talents in Universityof Ministry of Education of China (No. NCET-11-0766).

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7

1. Introduction2. Materials and methods2.1. Diatom frustules preparation2.2. Fabrication of the C. argusN. soratensis frustule package array2.3. Fluorescent antibody detection

3. Results and discussions3.1. Morphology of diatom frustules3.2. Package array of C. argusN. soratensis3.3. Fluorescent antibody detection results3.4. Potential and future plan

4. ConclusionsAcknowledgmentsReferences