微液滴中的基因检测

Au nanoparticles enhanced fluorescence detection of DNA hybridization in picoliter microfluidic droplets

Hongwei Zhu &Guodong Wang &Donglei Xie &Bo Cai &Yumin Liu &Xingzhong Zhao

Published online:7March 2014

#Springer Science+Business Media New York 2014

Abstract This work reports a facile microfluidic device for Au-nanoparticle enhanced fluorescence detection of tiny amount of nucleotides within droplets in a high-throughput way.Droplets containing single strand DNA probe and rele-vant complementary strands DNA(cDNA)are generated in flow-focusing manner and the hybridization between them is realized in droplets flowing along a long serpentine channel.In order to find the optimal experimental condition,finite element method simulation is used to predict the interface evolution between the two phase liquids.Based on the fluo-rescence emited by intercalator reacted with the generated double-strand DNA(dsDNA),the target cDNAwith a concen-tration of 1nM can be detected in droplets.And when we adopt Au nanoparticles to immobilize DNA probe which can amplify the fluorescence intensity,10pM completary DNA could be detected.Due to the advantages in high-throughput and compartmentalization of this droplet platform,the detec-tion procedure can be finished in 3h.Our method shows good potential application in facile,sensitive,low cost and fast DNA detection for applications in personal health care.Keywords Microfluidic droplets .DNA hybridization .Gold nanoparticles .Fluorescence detection

1Introduction

Since human genome project has been completed in 2003,exploring a cheap,fast,and simple DNA syquencing method has become a hot topic (Boles et al.2011;Khandurina et al.2000).The detection and quantification of DNA hybridization is greatly important in genotyping,medical diagnostics,path-ogen detection and forensic science (Ben-Yoav et al.2012;Dong et al.2010;Wilhelm 2009).Numerous methods includ-ing microarray (Servoli et al.2012),quartz crystal microbal-ance (Chen et al.2009),field effect transistor (Kim et al.2011),and surface plasmon resonance (Acuna et al.2012)have been developed to analyze DNA hybridization,sequenc-ing and single nucleotide polymorphism(SNP).In all these methods,DNA detection is often carried out with sensing DNA probe immobilized on a substrate surface to capture and sense DNA target in bulk solution.Which however faces several challenges.First,target molecules toward the DNA sensing probes are conducted by passive diffusion which greatly hinders the hybridization rate.Thus,it usually takes from several hours to overnight to complete DNA detection.Second,the random probe immobilization density,as well as electrostatic hindrance and length of DNA not only affect the efficiency of DNA hybridization and sensing,but also account for the poor repeatability of experiments.How to deal with these problems and develop a fast,more efficient,more accu-rate,and less sample consuming technique for DNA analysis is a worthwhile topic.

Droplet-based microfluidic is becoming a powerful method for drug discovery,biological assays,and DNA hybridization attributed to its key characteristics of compartmentalization,integration,quick reaction,and versatility (Zhu et al.2011;Link et al.2006).Rapid mixing and reaction of fluids can be achieved in microfluidic droplets.And the droplets composi-tion,motion,volume and generation frequency are controlla-ble with the modulation of flow rates of immiscible carrier

Electronic supplementary material The online version of this article (doi:10.1007/s10544-014-9850-8)contains supplementary material,which is available to authorized users.

H.Zhu (*):G.Wang :D.Xie

School of Electrical Engineering and Automation,Henan Polytechnic University,Jiaozuo 454003,China e-mail:zhwgo@https://www.360docs.net/doc/bc7198421.html,

H.Zhu :B.Cai :Y .Liu :X.Zhao

Key Laboratory of Artificial Micro-and Nano-Structures of Ministry of Education,School of Physics and Technology,Wuhan University,Wuhan 430072,China

Biomed Microdevices (2014)16:479–485DOI 10.1007/s10544-014-9850-8

fluids and disperse fluids.DNA detection in picoliter microfluidic droplets are already reported(Hsieh et al. 2009);(Hadorn et al.2012;Salm et al.2013;Boles et al. 2011;Sochol et al.2011;Cecchini et al.2011).And the digital polymerase chain reaction for detection and quantitation of specific nucleic acid sequences are also implemented in water-in-oil droplets of nanoliter volumes(Hindson et al.2011; McDermott et al.2013;Pinheiro et al.2011).Multiplexing dPCR in picolitre droplets with emulsions is reported(Zhong et al.2011).In these methods,detecting the fluorescence emission from DNA-binding dyes is a most popular method for DNA analysis(McDermott et al.2013;Zhong et al.2011).

It is reported that metal nanoparticles can achieve fluores-cence enhancement to improve the detection limit(Yeh et al. 2012;Tam et al.2007;Fu et al.2010).Au nanoparticles (AuNPs)are already used to improve the fluorescence inten-sity and become increasingly attractive tool for DNA detec-tion because of their size-depedant optical properties(Jans and Huo2012;Riskin et al.2010).And the AuNPs can overcome the poor sensitivity,poor stability,and safety problems asso-ciated with the use of radioisotopic,fluorescent,and enzyme label(Qi et al.2009).

Here we report a droplets based microfluidic plarform for DNA detection.In our device,DNA samples are encapsulated into droplets in flow-focusing manner and gradually hybridize to double strands along a serpentine channel.Based the fluo-rescence emitted by intercalator reacted with the generated dsDNA,target cDNA in droplets can be detected.And we tried to quantify dsDNA by recording the changes in fluores-cence intensity.For the purpose of improving sensitivity,Au nanoparticles are utilized to immobilize DNA probe and en-hance the fluorescence intensity.At last,10pM cDNA hybrid-ization is detected with this method.And the whole experi-ments could be accompleted in3h.Our approach shows good potential in facile,fast and sensitive DNA detection for appli-cation in point of personal health care.

2Experiment

2.1Reagents and materials

Reagents and solvents were acquired from commercial sources and used as received without further purification. We bought soybean oil from Beiya Medical Oil Co.Ltd., China.Polydimethylsiloxane(PDMS,RTV615)from GEToshiba Silicones Co.Ltd was used to fabricate microfluidic chip.Chloroauric acid,bovine serum albumin and trisodium citrate were obtained from Sinopharm Chemical Reagent Co.,Ltd.Sorbitan monooleate(Span80) was purchased from China National Medicines Corporation. The fluorescent intercalator SYBR@Green was purchased from Invitrogen.Photoresist SU8-2050was purchased from Clariant Corp.Phosphate buffer solution acquired from Hyclone Corp was utilized to dilute DNA sample.The DNA oligonucleotides were purchased from Integrated DNA Technologies(Bhattacharyya et al.2012),Inc.The sequence is as follows:

single strand DNA(ssDNA)probe:5′-end(5′-/5ThioMC6-D/ GTA AAA CGA CGG CCA G-3′)

complementary DNA(cDNA):5′-CTGGCCGTCGTTTT AC-3′

non-complementary DNA(ncDNA):5′-CAGGAAAC AGCTTGAC-3′

2.2Chip design and fabrication

The microfluidic device consists of two parts:the flow-focusing channel and the serpentine channel,just as shown in Fig.1.First,droplets containing ssDNA probe and cDNA are generated in flow-focusing orifice and then transported down to the serpentine channel.It takes a long time for the droplets travelling in the serpentine channel.That provides sufficient time for hybridization between ssDNA and cDNA.

The device was fabricated with soft lithography technique. Photoresist SU8-2050was spincoated onto a4in.silicon wafer in thickness of50μm.The photoresist layer was ex-posed to UV light through a mask and developped to gain the mold.Then PDMS(precursor A and crosslinker B with a ratio of10:1)is degassed and casted against the photoresist mask to form the fluidic channel.After baked at75°C overnight,the PDMS layer was peeled off and bonded onto a galss substrate with oxygen plasma treatment(Harrick Scientific Corporation,USA).At last,the microfluidic device was baked for another3days at75°C to strengthen the bonding durability.

2.3Simulation

In order to find the optimal droplet generation condition,finite element method simulation is used to predict the interface evolution between the two immiscible liquids in flow-focusing orifice.We used COMSOL software to conduct a 2D simulation to optimize the condition for monodisperse droplets generation.The mass and momentum balances was modeled by incompressible Navier–Stokes equations.The interface between water phase and oil phase was described by level set function.The interfacial tension between fluids was measured with KRUSS interfacial tension tester(DSA 100Drop Shape Analyzer,KRUSS GmbH,Hamburg, Germany).The viscosity of soybean oil was measured by the Rheometrics ARES(TA Instruments Inc.).The water contact angle for the channel was set72°.The specific param-eters used in the calculation are shown in Table1.

2.4Experiment procedure

First,the channel was pretreated by 0.5%bovine serum albumin solution for 1h to minimize the reagent adsorption.The fluids were injected by syringe pumps (TS-60,Longer Precision Pump Co.Ltd,China)through poly (tetrafluoroethylene)(PTPE)tubes.5μM DNA probe solu-tion and cDNA solution were injected into the two inlets of the Y-shape channel individually,as shown in Fig.1a and b .Oil with 0.5wt%surfactant Span 80was injected to the inlet C as continuous phase.The A stream (ssDNA probe)and B stream (cDNA)could be mixed and then dispersed in continuous phase of oil.Therefore,monodisperse Droplets containing ssDNA and cDNA were initially produced in the upper flow-focusing geometry.Then droplets would get to the ser-pentine channel,where hybridization began.For observation of the DNA hybridization,fluorescent intercalator SYBR @Green was added into the cDNA solution with the ratio of 1:400(SYBR @Green :cDNA).The hybridization of DNA can be confirmed by observing the fluorescence emited by SYBR @Green labling double strands DNA (dsDNA)un-der the irradiation of mercury lamp.

The droplets based DNA hybridization method can be used to detect different concentration of cDNA.And we used gold nanoparticles (AuNPs)to enhance the fluorescence intensity.AuNPs were synthesized by the reported method (Liu et al.2012).0.019g HAuCl4·3H2O was dissolved into 100mL deionized water.When the solution was heated to boiling,6.6mL of 0.066g sodium citrate solution was added into the boiling solution and the mixed solution would turn into rose color gradually.After that,the solution was cooled down

at room temperature.Then the resulted solution was centri-fuged at 12,000rpm for 15mins.After abandoning the super-natant,the AuNPs were redispersed in 96mL aqueous solu-tion.The obtained solution which contains 0.5mM AuNPs were mixed with ssDNA probe with a volume ratio of 1:5(AuNP :ssDNA probe).The gold nanoparticles capture DNA probe with covalently Au-SH bonding which makes the probe surround the AuNPs.The prepared solution B of differ-ent cDNA concentrations were injected into microfluidic channel in succession and individually.After solution of cDNA with different concentration injected,it took several minites for the droplets generation and DNA hybirdization in the serpentine channel.And the fluorescence intensity at the outlet was recorded to detect cDNA.The same processes were also conducted for non-complementary DNA with a concen-tration of 1μM for a control experiment.

The whole process was recorded with a high-speed CCD camera (DP72,Olympus,Japan)mounted on an inverted optical microscope (IX71,Olympus,Japan).A mercury lamp was used to excite the green fluorescence of SYBR @Green.All the images are processed by the Image analysis software (Imaging Pro Plus 6.0,Media cybernetics,Inc.).

3Results and discussion

3.1Generation of droplets containing DNA samples In the simulation,three different cases are observed as shown in Fig.2.Case A:the continuous phase is flowing slowly (0.1cm/s)and water phase is flowing quickly (0.5

cm/s).

Fig.1Schematic illustration of the microfluidic device.Solutions containing DNA probe and cDNA strand are individually injected into the two inlets of the Y shape channel:A and B .The aqueous stream is sheared by two oil streams in the orifice.Then the produced droplets containing DNA samples began to hybridize in the serpentine geometry.‘A ’is the inlet of DNA probe,‘B ’is the inlet of cDNA,‘C ’is the inlet of Oil phase,‘D ’is the outlet of solution,‘E ’is the observing point at the outlet

Table 1Parameters used in the simulation

Density of solution Solution viscosity Density of oil Oil viscosity Interfacial tension 1000(Kg/m 3)

1e-3(Pa ?s )

880(Kg/m3)

7.5e-2(Pa ?s )

3.2e-3(N/m)

Because of the hydrophilic feature of the channel,the water phase adhere to the surface and it ’s hard to generate droplets.And the numberical stability is not very good at the same time.Case B:the disperse phase flows quickly (0.5cm/s)and oil quickly (0.5cm/s).Two phase fluids display laminar flow phenomenon.The continuous phase of oil can ’t shear the water streams into disperse droplets.Case C:disperse phase flows slowly (0.1cm/s)and oil phase flows quickly (0.5cm/s).The water droplets are produced.As seen from Fig.2,there are small satellite droplets generated simultaneously.It is difficult to produce highly monodisperse droplets.Through calculation,the droplets can be generated at the condition with disperse speed at 20~30μL/h,while oil speed at about 100μL/h.In order to generate highly uniform DNA droplets,we set the flowing velocity of water phase at 30μL/h and adjust the flow velocity of oil phase at the range of 40~130μL/h in experiments.Process of droplets formation can be observed in the bright field.The statistics of prepared droplets was displayed in Table 2.

From Table 2,we can get that when the aqueous solution flows at 30μL/h and oil at 60μL/h,highly monodisperse droplets can be produced.So that,we adopt these parameters to generate micro-droplets containing DNA samples for sub-sequent experiments.

3.2DNA hybridization in droplets

When the droplets entered into the supertine channel,cDNA became to hybridize with the ssDNA probe.Figure 3was a bright-field picture of the droplets flowing in serpentine chan-nel.From it,we can see that droplets was uniform-sized and evenly spaced.Each of them provides an idea reaction cham-ber for DNA hybridization (Horsman et al.2007).As droplets flowed down and the hybridization proceeded,more double strands DNA (dsDNA)were generated which could afford more fluorescent dye.As the result,the fluorescence intensity is getting brighter.The more DNA hybridized,the stronger fluorescence intensity became.So the amount of DNA hy-bridization can be characterized with the fluorescence intensity.

In order to have a clear observation of the hybridization process,four pictures in Fig.4were taken to record fluores-cence intensity at different places of the serpentine channel.Figure 4a was taken near the inlet of serpentine channel,Fig.4b was taken at the near left area,Fig.4c was at the near right area,and Fig.4d near the outlet.In Fig.4a ,the fluores-cence was weak at the starting area.And we could see that DNA hybridization was started at the inlet of serpentine channel.As the droplets flowed downstairs,fluorescence in-tensity was getting stronger and stronger because of hybridi-zation.In Fig.4b ,the intensity was significantly enhanced compared with Fig.4a .And in Fig.4c ,fluorescence intensity was further enhanced.In Fig.4d ,the strongest

fluorescence

Fig.2Three situations of droplets generation in the simulation.Condi-tion a :The aqueous phase is adhere to channel wall and droplets can ’t be acquired when the continuous phase has a low velocity and the disperse phase has a high velocity.Condition b :the fluids demonstrate a laminar condition with both the continuous phase and the disperse phase have a high flowing velocity.Condition c :droplets are obtained as oil phase flows quickly and disperse phase flow slowly

Table 2The relationship between dimentions of droplets and flow rates of oil

Oil velocity (μL/h)Average diameter (μm)Standard deviation (μm)40172 3.350153 2.160131 1.070105 1.59089 2.011071 2.5130

60

3.4

Fig.3Droplets containing DNA samples flow in the serpentine channel.The scale bar is 200μm

intensity was observed.At the outlet,we could also see that fluorescence intensity was getting saturated,which indicates that DNA hybridization was almost completed.For the non-complementary DNA interaction with DNA probe,two pic-tures were taken at the inlet and outlet of serpentine channel.The pictures were presented in Supporting information .We

could see that fluorescence intensity at the outlet was almost the same as at the inlet.It could be deduced that the non-specific interactions between ssDNA probe and ncDNA did not affect the fluorescence intensity a lot.

In order to quantitatively analyze DNA hybridization pro-cess,we measured the fluorescence intensity along

the

Fig.4The fluorescence intensity in serpentine channel resulted from hybridization of DNA in droplets.a is fluorescence intensity in the inlet part of

serpentine channel,b is near left part of serpentine channel,c is near right part,and d is at the outlet.The red scale bar is 200μ

m

Fig.5Increasement of the fluorescence intensity along the serpentine channel.With the DNA hybridization going,

fluorescence intensity is getting brighter.And the higher the concentration of cDNA,the brighter the fluorescence

intensity.The bottom curve is the fluorescence intensity

increasement by the interaction between ncDNA and DNA probe

serpentine channels.Fluorescence intensity statistical result was shown in Fig.5.As the increasement of concentration of cDNA,the generated fluorescence intensity were getting brighter and brighter.For the ncDNA interaction with DNA probe,the fluorescence intensity was not getting much brighter which means nonspecific interaction didn ’t have an important effect on the experiment result.It also could be seen that the curve was steeper at first and then gradually became more flat.This gave us a hint:at the early stage,DNA hybridization happened quickly.But as the hybridization proceeded,it was getting slower.The fluorescence intensity became brightest and saturated when the droplets arrived at the outlet.It indi-cated that the hybridization was almost completed at the outlet.In the following experiments,an observing point E was settled at the outlet for cDNA detection.

3.3Au nanoparticles enhanced DNA hybridization for cDNA detection

The morphology of produced Au NPs was observed by transmission electron microscopy.The optical absorption spectroscopy measurements were conducted with a UV –vis spectrophotometer (Cary 5000,Varian).In Fig.6a ,AuNPs are about 15nm in size.From Fig.6b ,it can be seen that the spectrum adsorption peak was near 540nm.The AuNPs are used to immobilize DNA probe to detect cDNA.And for the control experiment,cDNA detection is also car-ried out by DNA probe without immobilization of Aunano particles.Figure 7shows the Aunano particles enhanced fluo-rescence detection of cDNA at different concentrations.We found that the fluorescence intensity is brighter with Aunano particles surrounded by DNA probes than without Aunano particles.More importantly,we can detect 10pM cDNAwhich was two orders of magnitude better than without Aunano particles which could detect 1nM cDNA.The performance is better because AuNPs can enhance the fluorescence inten-sity.First,The thiolated ssDNA probe is linked to Aunano particles through a covalent bonding (Au-SH).After

hybridization,dsDNA which labelled with fluorescence mo-leculars are captured around gold nanoparticles which cause the aggregation of fluorescence.And the aggregation of fluo-rescence can improve the fluorescence intensity significantly.Second,the emitted fluorescence of YBR @Green covers the spectrum of 540nm,which is consistent with the spectrum adsorption peak of Aunano particles.When the emited fluo-rescence gets on the surface of AuNPs and contacts with the electrons of Au nanoparticles,the plasmon resonance phe-nomenon occurs which could enhance the fluorescence inten-sity (Wang et al.2010;Lakowicz et al.2008).On the one hand,metal nanoparticles improve the coupling efficiency of the fluorescence emission to far-field through scattering.On the other hand,the optical absorption and scattering properties of metallic nanostructures can adjust the radiative decay rate and direction of fluorophore emition which can enhance the near-field optical intensity.These reasons make the generated fluorescence intensity

brighter.

Fig.6Characterization of gold nanoparticles.a TEM micrograph of produced Au nanoparticles.b UV Spectrum adsorption of gold

nanoparticles

Fig.7Fluorescence detection of cDNAwithin droplets.Without Aunano particles,1nM cDNA could be detected.And the standard deviation of fluorescence intensity was about 09.With Aunano particles,as low as 10pM cDNA hybridization could be detected and the standard deviation was reduced to 0.75

From Fig.7,we can also see that with increasing of cDNA concentration,fluorescence intensity is getting stronger and stronger.And Because of the fast DNA hybridization speed in droplets,the experiment is done in a short time.One concen-tration of cDNA solution injected into microfluidic channel, the hybridization process can be finished in a quarter of an hour.And the whole experiment could be finished in3h.

4Conclusion

Here a facile method of DNA detection is demonstrated with the sensitive and high-throughput droplet-based microfluidic platform.For producing highly monodisperse droplets con-taining DNA samples,finite element method simulation is employed to forecast the formation of droplets.Single strand DNA probe and relevant cDNA are encapsulated in droplets in the flow-focusing part.After droplets flowed into the ser-pentine channel,DNA probe began to hybridize with cDNA gradually.As the proceeding of hybridization goes on,fluo-rescence intensity is getting stronger and stronger.Continuous observation of DNA hybridization process is achieved by recording the fluorescence intensity.By recording the emited fluorescence,cDNA is detected.To improve the detection limit,Au nanoparticles are used to immobilize DNA probe and improve the fuorescence intensity.At last,10pM cDNA can be detected within massive amount droplets.Due to advantages in high-throughput and compartmentalization of this droplet platform,the detection could finish within3h. This approach shows good potential in facile,fast and sensi-tive DNA detection for applications in point of personal health care.

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