dCas9-mediated Nanoelectrokinetic Direct Detection of Target Gene

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dCas9-mediated Nano-electrohydrodynamic Direct Detection of Target Gene for Liquid Biopsy Hyomin Lee, Jihye Choi, euihwan Jeong, Seongho Baek, Hee Chan Kim, JongHee Chae, Youngil Koh, Sang Woo Seo, Jin-Soo Kim, and Sung Jae Kim Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b03224 • Publication Date (Web): 13 Nov 2018 Downloaded from http://pubs.acs.org on November 14, 2018

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dCas9-mediated Nano-electrohydrodynamic Direct Detection of Target Gene for Liquid Biopsy Hyomin Lee1,*,+, Jihye Choi2, +, Euihwan Jeong3,4+, Seongho Baek2, Hee Chan Kim5, Jong-Hee Chae6, Youngil Koh7, Sang Woo Seo8, Jin-Soo Kim3,4* and Sung Jae Kim2,9,10,*

1

Department of Chemical & Biological Engineering,

Jeju National University, Jeju, 63243, Republic of Korea, 2

Department of Electrical and Computer Engineering,

Seoul National University, Seoul 08826, Republic of Korea 3 4 5

Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea,

Center for Genome Engineering, Institute for Basic Science, Seoul 34047, Republic of Korea,

Department of Biomedical Engineering, Seoul National University, Seoul 08826, Republic of Korea, 6

Department of Pediatrics, Seoul National University, Seoul 08826, Republic of Korea, 7

Department of Internal Medicine, Seoul National University Hospital, Seoul 03080, Republic of Korea,

8

School of Chemical and Biological Engineering, Institute of Chemical Process, Seoul National University, Seoul 08826, Republic of Korea,

9

Nano System Institute, Seoul National University, Seoul 08826, Republic of Korea 10

Inter-university Semiconductor Research Center,

Seoul National University, Seoul 08826, Republic of Korea

1

ACS Paragon Plus Environment

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+

These authors contributed equally. Correspondence should be addressed to Prof. Sung Jae Kim, Prof. Jin-Soo Kim and Prof. Hyomin Lee: E-mail: (SJKim) [email protected]; phone: +82-2-880-1665, (JSKim) [email protected]; phone: +82-2-880-9327, (HLee) [email protected];

*

2


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Abstract The-state-of-the-art bio- and nanotechnology have opened up an avenue to non-invasive liquid biopsy for identifying diseases from biomolecules in bloodstream, especially DNA. In this work, we combined sequence-specific-labeling scheme using mutated Clustered Regularly Interspaced Short Palindromic Repeats associated protein 9 without endonuclease activity (CRISPR/dCas9) and ion concentration polarization (ICP) phenomenon as a mechanism to selectively preconcentrate targeted DNA molecules for rapid and direct detection. Theoretical analysis on ICP phenomenon figured out a critical mobility, elucidating two distinguishable concentrating behaviors near a nanojunction; a stacking and a propagating behavior. Through the modulation of the critical mobility to shift those behaviors, the C-C chemokine receptor type 5 (CCR5) sequences were optically detected without PCR amplification. Conclusively, the proposed dCas9-mediated genetic detection methodology based on ICP would provide rapid and accurate micro/nanofluidic platform of liquid biopsies for disease diagnostics.

Keywords Ion

concentration

polarization,

selective

preconcentration,

micro/nanofluidics, direct detection

3


dCas9,

liquid

biopsy,

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Ever-increasing interests in point-of-care diagnostics have thrived for the detection of targeted genetic materials over the past decades due to the demands for early stage diagnosis. Various detection methods have been refined based on multidisciplinary fundamentals and engineering tools such as Sanger sequencing1, cyclic array sequencing2, next generation sequencing (NGS)3, etc. Advances in these state-of-the-art technologies have opened up a new avenue to liquid biopsies. These non-invasive tools provide detailed spectrum of disease progression, which is rarely obtained by invasive tissue-based biopsies, through analyzing floating bio-samples in bloodstream such as circulating tumor cell, circulating tumor DNA, tumor-derived exosomes, etc4. Most of these works include the replication of nested sets of DNA fragments by polymerase chain reaction (PCR) due to low abundant targeted sequences at the early stage of disease progression5. Electrophoretic separation is required then to determine the order of the whole sequence. In general, the separation resolution is governed by the difference of the electrophoretic mobility between the fragments. The mobility () is proportional to the ratio of a fragment’s net charge (qn) to the friction coefficient (Cf) of it (i.e. DNA ~ qn / Cf). Since DNA molecule behaves as a free-draining coil instead of an impermeable rigid sphere6, 7, DNA fragments over 50 base pairs have no difference in their mobility8 so that the separation peaks have become undistinguishable in free solution. To address the issue, gel electrophoresis and end-labeled free-solution electrophoresis have been introduced to physically modify the mobility of DNA molecules based on their size9, 10. Although a myriad of incredible biological discoveries have been provoked by PCR process with the separation methods1, those are timeconsuming and labor-intensive since these methods are based on non-specific labelling and base-by-base full sequencing. Additionally, amplification by PCR itself has unavoidable

4


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errors11 and repeated sequences abundant in diseases, especially cancer, could remarkably reduce diagnostic accuracy 12. To resolve the problem in PCR amplification, researchers have demonstrated direct nanopore sensing13,

14

and ionic-field-effect-transistor sensor15,

16

. For the former, the intra-channel

segment of the translocating molecule causes an effective conductance alteration so that the statistical analysis of dwell time and current drop realizes the detection of target sample. For the latter, the targeted DNA/RNA passing through cis- to trans-chamber is immobilized on the functionalized nanopore surface, leading the capacitance of oxide layer between the fluidic channel and gate electrode to change. The existence of the target molecules can be confirmed by measuring the oxide capacitance. Although these methods can detect targets without PCR amplification, they possess inherent low signal-to-noise ratio, extremely short dwell time to distinguish the translocating molecules and expensive fabrication processes. Instead of these sophisticated sensors, ion concentration polarization (ICP) phenomenon near a charge-selective nanojunction was employed in this work. Due to the overlap of electrical double layers on the interior walls of the nanojunction17, 18, only counter-ion can pass through the nanoscale pore while co-ion cannot. Around the interface of bulk electrolyte and the nanojunction, the charge-selective transport generates an ion concentration boundary layer where there exist the multiscale electro-physicochemical-hydrodynamic couplings of locally amplified electric field19,

20

, extremely low ion concentration21-23, non-equilibrium space

charge24-26, ionic hydration shell stripping27, electroconvection28-30 and associated overlimiting currents31-33. With the aid of the ICP phenomenon, DNA34, 35, proteins36, 37 and cells38 have been reported to be concentrated over a detectable level and the concentrating mechanism is balanced between electrophoretic force and convective drag force39-42 as shown in Figure 1(a). Since the preconcentration is solely driven by the physical force balance, the detection can be 5


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free from the inevitable errors caused by PCR amplification. The fluorescence-labeled concentrated samples can also be optically observed, leading to the direct determination of the presence of target sequences. The separation of targeted DNA from non-targets can be achieved by employing a sequence-specific DNA-binding protein, dCas943 (catalytically inactive Clustered Regularly Interspaced Short Palindromic Repeats associated protein 9 (CRISPR/Cas9)44). The detection protocol for target sequences using Cas9 has been recently reported that particular genomic loci are cleaved and then the related changes of gene expression are observed indirectly45. The indirect method using Cas9 should require additional sensing of altered gene expression but the dCas9-mediated method with ICP would provide straightforward information on the target DNA, attributed to the sequence-specific binding and selective preconcentration. In this work, we suggest a dCas9-mediated nano-electrohydrodynamic direct detection platform based on the sequence-specific dCas9-capturing mechanism and the ICP phenomenon (Figure 1(b)), which was verified by optically detecting a synthesized C-C chemokine receptor type 5 (CCR5) gene related to human immunodeficiency virus (HIV) as a disease model. First, a criterion between two distinct preconcentration patterns as (i) stacking and (ii) propagating behavior was analytically derived by defining a critical mobility obtained from the electrohydrodynamic balance of charged species near the concentration boundary layer. Adjusting the tunable critical mobility, dCas9-DNA complex was kept to be a stacking behavior in the vicinity of nanojunction, while free DNA had a propagating behavior toward bulk reservoir, leading to simultaneous separation and preconcentration of the targets from nontargets. As a result, we successfully detected the optical signals of each analyte without PCR as schematically represented in Figure 1(c). This methodology would enable the direct recognition of disease-related sequences as an effective point-of-care platform. 6


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Analysis of nano-electrohydrodynamics near nanojunction. In general, the governing equations of fully-developed ICP phenomenon were the Poisson equation, the Nernst-Planck equations, the continuity equation and the Stokes equations as following.

 E  zF  c  c  ,

(1)

j   D c    c E  c u and   j  0,

(2)

0  p 2u  zF  c  c  E and  u  0

(3)

where  the electric permittivity of water, E the electric fields, z the ion valence for z:z symmetric electrolyte, F the Faraday constant, c+ and c- cation and anion concentration, j± the ionic flux of each ion, D± the diffusivity of each ion, ± the mobility of each ion (in this work, the mobilities of both cation and anion were treated as positive value for convenience), u the flow fields, p the pressure and  the viscosity of water. The numerical solutions were obtained using commercial software (COMSOL Multiphysics 4.4) with appropriate boundary conditions of (i) specific potential, bulk electrolyte concentration, hydrodynamic open boundary at each reservoir, (ii) the Donnan potential, the Donnan concentration for cation, no-flux condition for anion, no-slip condition at the surface of nanojunction and (iii) no penetration conditions for electric field and each ionic species, electroosmotic-slip condition with zeta potential of -2.5 mV at the microchannel walls. Note that the interior of nanojunction and the cathodic microchannel were excluded from the numerical domain because the negatively charged analyte was mainly accumulated at the anodic microchannel. Supporting Information, Note 1 showed the results of the spatial concentration in the 2D domain, area-averaging concentration profile along the x-direction and area-averaging electric field strength along the x-direction obtained by the fully-coupled 2D numerical simulations. Due to charge-selective transport through the nanojunction, the ion depletion zone denoted in those figures was developed near 7


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the interface, leading the electric field to be locally amplified inside the zone, as in experimental measurements in previous literature19. Since the amplified electric field acts as a virtual barrier to a negatively charged analyte, the region outside the ion depletion zone has been utilized to concentrate charged molecules31, 34, 36, 46, 47. The ICP phenomenon in this platform was strongly contributed by the surface conduction32, 33, 38, 48, 49

. The electroosmosis was weak mechanism here and the electroconvective instability

was negligible due to the highly-confined microchannel. The analysis of dominant factor on ICP can be found in Supporting Information, Note 2. Under this situation, the 3D actual domain can be analyzed in lower dimension (2D or 1D) without losing the important characteristics of ICP. Previous experiments48, 50, 51 with this confined device also showed 2D-like flow fields which support the validity of our lower dimension analysis. In order to obtain analytical solutions, the governing equations were converted into an analytically tractable problem based on the area-averaging model27 under following assumptions in the domain; (i) local electroneutrality (i.e. zc+ - zc- + zAcA = 0 where zA and cA the valence and the local concentration of an analyte, respectively) and (ii) extremely low analyte concentration (i.e. cA L* as shown in Supporting Information, Note 1, demonstrating agreement regardless of voltage configuration.

Critical mobility to determine micro/nanofluidic preconcentration behaviors. The flux of

a negatively charged analyte is expressed as

j A   DA

dc A   A c A E x  c Au x dx

(8)

where jA the area-averaging analyte flux, DA the analyte diffusivity, cA the analyte concentration and A the electrophoretic mobility of analyte. If the diffusive transport is negligible through the microchannel, the concentrating behaviors of the analyte can be determined by comparing 9


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the scale of electrophoretic migration and convective transport. When the electrophoretic migration is larger than the convective transport, the preconcentrated analyte would consistently move toward the bulk reservoir and the plug finally escapes the microchannel. On the other hand, the charged analyte is translated with fluid flow toward the nanojunction when the convective transport is larger than the electrophoretic migration. However, since the amplified electric field inside the ion depletion zone rejects the analyte to pass through, the charged analyte would be preconcentrated at a fixed position, where jA = 0, inside the microchannel. If the fixed position is set to be at the entrance of microchannel, following equality jA

xL

  cr c A Ex

xL

 cA ux

xL

0

(9)

should be held at x = L. We defined the analyte mobility at this situation as a critical mobility (cr) which satisfies the equation (9). The combination of the equations (5) and (7) to (9) with L >> L* results in   QL   exp     D A   cr D  D eff  I      zFQc0 Deff     QL     1  exp    D A    eff   

1

(10)

which was the closed form of the critical mobility. Utilizing this criterion, we can predict how the analyte behaves near the ion depletion zone. Langevin dynamics simulations were conducted as shown in Figure 2(a) and 2(b) using customized C program. The analyte was concentrated at a fixed position as shown in Figure 2(a) when the analyte mobility was smaller than the critical value (A = 0.9 cr). Meanwhile, Figure 2(b) showed the case of A = 1.1 cr, where the electrophoretic migration of the analyte was larger than the convective transport so that the analyte eventually came out of the 10


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microchannel. To classify these two behaviors, the cases of A < cr and A > cr were named as “a stacking behavior” and “a propagating behavior”, respectively. Two distinguishable behaviors were experimentally verified as well in Figure 2(c) and 2(d). The fabrication of the micro/nanofluidic device and experimental setup were described in Supporting Information, Note 3 and Supporting Information, Note 4, respectively. In the experiments using KCl electrolyte, the parameters in equation (10) were L = 7 × 10-3 m, A = 2.25 × 10-9 m2, I = 1.12 × 10-7 A, Q = 5.32 × 10-13 m3/s, c0 = 1 mol/m3 so that cr = 2.7 × 10-8 m2/V/s. The mobility of sulforhodamine B (1.26 × 10-8 m2/V/s) 53 was smaller than cr, while the mobility of Alexa 488 (3.61 × 10-8 m2/V/s) 54 was larger than cr. Thus, sulforhodamine B and Alexa 488 were preconcentrated showing a different behavior according to the critical mobility-based analysis. See Supplementary Video 1. In the simulation results of Figure 2(a), the analyte was preconcentrated at the boundary of the depletion zone. On the other hand, the analyte in the experimental results of Figure 2(c) was preconcentrated at the boundary of the depletion zone but the preconcentrated plug extended towards a reservoir. Such expansion was not the result of propagating behavior; it is attributed to the interaction between concentrated analyte and background electrokinetic force field. From the previous numerical study 55, the locally accumulated analyte would alter the electrolyte concentration profile, electric field and flow field near the nanojunction. However, it is non-feasible to describe such interaction of analyte species by our simple 1D model. Additionally, our Langevin dynamics simulations neglected the complex interactions to simply verify the concentrating behavior depending on cr. Nevertheless, the force field alteration cannot lead to the transition of concentrating behavior (e.g. from stacking to propagating pattern or vice versa). 11


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The most eye-catching feature of cr is that it has not only intrinsic parameters (diffusivity and mobility of electrolytes) but also extrinsic parameters (ionic current (I), electrolyte concentration (c0), flow rate (Q) and microchannel dimensions (L, A)). Therefore, the concentrating behavior can be switched by controlling these parameters at one’s discretion. (See Supporting Information, Note 5.) The analytical tractability of cr enabled the selective preconcentration of target sequence.

Nano-electrohydrodynamic on-chip detection of target gene with dCas9. We estimated the

electrophoretic mobility of RNA-guided dCas9 as a rigid sphere from the Henry’s equation52;

A=2A/3 f(aA) where A the zeta potential of charged analyte,  the inverse Debye length, aA the hydrodynamic radius of charged analyte and f(aA) represents the Henry’s function. ‘dCas9’ below denotes RNA-guided dCas9 for convenience (i.e. the word, dCas9, means the integrated dCas9 with guide RNA). The hydrodynamic radius of dCas9 was regarded as 6.5 nm56. Considering the crystal structure of RNA-guided Cas9, the tail of guide RNA exposed outside Cas9 took a small part (less than 0.2 v/v%) of the entire protein complex57. This small part of guide RNA was assumed to barely affect the diffusive motion of the entire protein, and thus, the effect of guide RNA on the increment of radius was not critically considered when it binds to virtually-spherical dCas9. On the other hand, the zeta potential of dCas9 was adopted as -20 mV as the previous study measured58. Using the hydrodynamic radius and the zeta potential of dCas9, the electrophoretic mobility of dCas9 was calculated as 1.09 × 10-8 m2/V/s. The electrophoretic mobility of 50 bp dsDNA is known as 3.62 × 10-8 m2/V/s8. In the case of dCas9-DNA complex, the alteration of total charge due to dCas9-binding was negligible in the scaling law of DNA mobility (DNA ~ qn / Cf) because the total charge of dCas9 was 160 times 12


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smaller than that of the 50 bp dsDNA59. In other words, the total charge of dCas9-DNA complex was approximately equal to that of 50 bp dsDNA. However, the hydrodynamic radius of dCas9 (6.5 nm) was comparable to the size of the DNA molecule (~ 11.2 nm)60 so that the increased hydrodynamic radius of dCas9-DNA complex resulted in the increment of friction coefficient, causing the significant decrease of the mobility by 36.7%. Consequently, the mobility of dCas9-DNA complex can be approximated as 2.29 × 10-8 m2/V/s. Therefore, if cr is set between 2.29 × 10-8 m2/V/s and 3.62 × 10-8 m2/V/s, the dCas9-DNA complex and free DNA would have different concentrating behaviors. To set such cr, we adopted parameters of L = 7 × 10-3 m, A = 2.25 × 10-9 m2, I = 1.12 × 10-7 A, Q = 5.32 × 10-13 m3/s, c0 = 1 mol/m3 and KCl electrolyte, leading cr to be 2.7 × 10-8 m2/V/s. The result of concentrating free DNA (synthesized CCR5 gene) labeled with FAM dye was shown in Figure 3(a). Due to the absence of Cy3-labeled dCas9, its concentrating plug was observable only by a green filter. The mobility of the free DNA (3.62 × 10-8 m2/V/s), which was larger than cr (2.7 × 10-8 m2/V/s), resulted in a propagating behavior. In contrast to the free DNA, dCas9 was preconcentrated in a fixed position near nanojunction because its mobility (1.09 × 10-8 m2/V/s) was lower than cr (Figure 3(b)). dCas9 labeled with Cy3 was only detectable by a red filter. The most important result for dCas9-DNA complex was shown in Figure 3(c). See Supplementary Video 2. The detailed preparation steps of dCas9-DNA complex can be found in Supporting Information, Note 6. The sequence-specific binding of dCas9 altered the electrophoretic mobility to 2.29 × 10-8 m2/V/s below cr so that a new stacking green plug appeared near nanojunction. We inferred that the newly appearing plug nearby the nanojunction was the dCas9-DNA complex because it was observed in both green and red filters. Considering the binding equilibrium, all of the DNA molecules were not able 13


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to react with dCas9 since the concentration of DNA and dCas9 was the same in our experiment. For this reason, the free DNA plug (i.e. dCas9-unbound DNA) still existed and had a propagating behavior even if it possessed the target sequences. If there was no sequencespecific binding, only propagating plug would appear. See Supporting Information, Note 7. In Figure 3(c), there are two green plugs and one could wonder if unbound DNA exists in the stacking plug (brighter one). As depicted in Figure 3(a), unbound DNA does not exist in a form of stacking plug within our parameter settings. Since DNA mobility is relatively high, higher flow rate should be required for this nano/microfluidic device to let the unbound DNA accumulate at the boundary of the depletion zone. Therefore, the newly appearing green plug in Figure 3(c), compared to Figure 3(a), only indicates the bound DNA, not the unbound DNA. The selection efficiency would be determined by the biological binding efficiency of dCas9 to the target DNA. When DNA targets are so low-abundant that erroneous PCR is prerequisite for detection, this ICP-based target DNA detection would become a suitable detection strategy without PCR. Since any negatively charged analyte is rejected to pass through the depletion zone, the analyte can be accumulated near the depletion boundary as long as its mobility is below cr. Consequently, the signal of dCas9-DNA complex would be electrohydrodynamically enhanced over an optically detectable level (~ 10 nM34). For the case of A < cr, the analyte influx can be approximated as JA = QcA0 where JA the influx and cA0 the bulk concentration of analyte. Under our experimental conditions, the influx of dCas9-DNA complex was about 7.05 × 10-20 mol/s. Such value corresponded with the concentrating rate of 1.88 M/min when the size of detection window was defined as 150 m width × 1 m length × 15 m depth. The experimentally measured preconcentration rate was discussed in Supporting Information, Note 14


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8. This implied that 627-fold preconcentration factor was able to be achieved within only 1 minute. Since the flow rate is, indeed, an adjustable factor, the concentrating rate can be further enhanced. For example, if the flow rate is adjusted from current value of 1.37 nL/min to 13.7 nL/min, then it ideally takes 100 minutes to detect 3 pM concentration of DNA (the concentrating rate is 18.4 pM/min), which is typical concentration of DNA from HIV positive cells withdrawal from infected patients61, 62. However, the limit of detection (LOD) in real world depends on both the preconcentration rate of targets and the binding affinity of dCas9. For instance, the sigmoidal binding curve of dCas9-DNA complex formation44 (i.e. the binding does not linearly proportional to the sample concentration) significantly limits LOD. Thus, For further study, it is required to biologically optimize binding reaction (such as regulating the optimal ratio of dCas9 and sgRNA, etc.) in order to enhance LOD. Nonetheless, our new detection mechanism is globally valid in any case of sensing based on mobility shift so that this mechanism will greatly benefit researchers in the field of biosensing, environmental sensing, etc.

Detection selectivity with respect to off-targets. The instant recognition of target sequence

has been newly attempted on the advent of sequence-specific binding materials such as Zinc finger transcription factors, transcription activator-like effector or aptamers63-66. Among the DNA-binding materials, Cas9 has received a huge limelight recently in the genome engineering field due to its highest target-specificity, the most efficient affinity to DNA and easy programmability66. We chose catalytically inactive Cas9 (dCas9) for the direct detection of target sequences rather than indirect detection by tracking the pathway of gene expression. The dCas9 possesses a programmable guide RNA and recognizes a protospacer-adjacent motif (PAM) on a DNA strand. Once dCas9 melts the DNA strands near the PAM, the base pairing 15


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between guide RNA and target sequence starts to propagate up to 20 base pairs. If the DNA sequence is not matched with the guide RNA within those pairs, the dCas9 is released from the DNA 43. Therefore 7 base pairs adjacent to the PAM, called a seed sequence, are known to be the most important parts for a primary reaction to recognize targets since the extensive pairing of dCas9 depends on the success of seed pairing66, 67. Therefore, it is necessary to confirm the selectivity of mismatches existing on target sequences for accurate diagnosis. In this work, artificial guide RNA molecules were introduced which were systematically mutated from a CCR5 guide sequence to own double-base mismatches at different positions 43, 44

. When mismatches between the guide sequence and the binding sequence occurred at 1, 2

positions, 6, 7 positions and 11, 12 positions far from the PAM, only one propagating plug appeared by a green filter as shown in Figure 4(a)-4(c), respectively. These observations implied that dCas9 recognized the mismatches on the binding sequence and did not bind to DNA molecules. However, in the case of mismatches on a PAM-distal region (19, 20 positions) as shown in Figure 4(d), a stacking and a propagating plug simultaneously appeared by a green filter. This was because the dCas9 failed to perceive the mismatches on the binding sequence and reacted with the off-targets similar to on-target. Namely, dCas9-DNA complex was formed although the DNA molecules were lack of an exact on-target sequence. These experimental results implied our method would be highly selective in the PAM-proximal region in accordance with the intrinsic characteristics of the dCas9-capturing process68. There exist certain clinical disorders with two consecutive base pair mismatches, where our test could be meaningful and useful for clinical diagnosis

69-72

. Nevertheless, it is worth

deliberating on the case for a single mismatch or double mismatches in separate places. The previous study by Qi et al. has shown that single mismatches in the seed region (the first 7nt from PAM) dramatically reduced repression activity of CRISPR/dCas9, caused by the loss of 16


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the complex-formation with target DNA67. It implies that the selectivity of our method can be determined by the fact that mismatches (either single or double at different locations) are present in the seed region. This is aligned with our experimental results with two contiguous mismatches. Thus, our method is able to selectively distinguish DNA molecules on the basis of whether mismatches (either a single or double at different locations) are present in this seed region or not.

Detection sensitivity with respect to DNA lengths. Our methodology to detect target

sequences is based on a mobility shift when free DNA are bound by dCas9.. The existence of targets was guaranteed for relatively short DNA molecules (50 and 100 bp) by observing both a stacking and propagating plug as shown in Figure 5(a). While the mobility of DNA was larger than cr before dCas9-binding (DNA was 1.34cr and 1.36cr each for 50 bp and 100 bp free DNA), it was shifted to become smaller than cr once dCas9 was bound to the DNA (complex was 0.85cr and 0.97cr each for 50 bp and 100 bp complex). However, in the case of relatively long target DNA (750 and 1023 bp), the physicochemical properties of the dCas9 could not be enough to alter the electrophoretic mobility of the long DNA remarkably as shown in Figure 5(b). Before dCas9-binding, the mobility of DNA was known to be DNA = 1.38cr and 1.39cr each for 750 bp and 1023 bp free DNA. In spite of dCas9-binding, only propagating plugs were observed with in these cases since the altered mobility due to dCas9-binding (complex was 1.21cr and 1.23cr each for 750 bp and 1023 bp complex) was still larger than cr and was approximately equal to that of free DNA as denoted in Figure 5(b). This is because the binding of single dCas9 to the relatively long DNA molecule was insufficient to change its friction coefficient and mobility to a great extent since the dCas9 was a small protein compared to the 17


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long DNA molecule. Therefore, the transition between the concentrating behaviors diminished and the dCas9-DNA complex remained in a propagating behavior. According to the scaling law of DNA mobility, it could be possible to detect long DNA targets by attaching multiple dCas9 proteins to several loci on the long DNA strand to effectively change the friction coefficient of them. The artificial fragmentation of DNA would be a possible remedy as well. It has been reported that the length of cell-free DNA molecules extracted from blood plasma has been known to be below 200 bp73 and DNA molecules from circulating-tumor cells are generally fragmented into 100 to 200 bp for sequencing. Thus, our suggestion as a dCas9-mediated platform to detect targeted DNA molecules would be an effective method to identify the presence of disease-related DNA molecules originated from cell-free DNA or cancer cells, etc.

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Ever-increasing desires for non-invasive and comprehensive disease diagnosis have demanded the emergence of advanced liquid biopsy tools on disease-related genetic sequences, viruses or cells. Here we presented a micro/nanofluidic platform for optical detection of targeted DNA sequences based on the sequence-specific dCas9-capturing mechanism and the ICP phenomenon. Through the rigorous fundamental analysis, we found the critical mobility (cr) to analytically classify the concentrating patterns near a nanojunction. When a negatively charged analyte had an electrophoretic mobility below the critical value, it was concentrated at a fixed position (i.e. a stacking behavior). On the other hand, the concentrating analyte eventually escaped the microchannel when its mobility was larger than cr (i.e. a propagating behavior). Adjusting cr by tweaking A, L, I or Q, etc. enables us to simultaneously preconcentrate and separate free DNA and dCas9-tagged DNA. In experimental demonstrations, we adopted synthesized CCR5 sequences which are related to HIV as a disease model. Setting the critical mobility to 2.7 × 10-8 m2/V/s, the dCas9-DNA complex (complex = 2.29 × 10-8 m2/V/s) was constrained to maintain the stacking behavior, while the free DNA (DNA = 3.62 × 10-8 m2/V/s) moved back to bulk reservoir in a propagating behavior. With the aid of ICP phenomenon, the signal of the targeted molecules would be efficiently enhanced over detection limit (627-fold/min). Thus, we were able to discriminate the presence of targeted sequences in direct and optical manner without PCR. Furthermore, the reliability of our detection methodology was experimentally confirmed with respect to off-targets and DNA length. Meanwhile, real samples in clinical practice have a variety of physiological conditions, calling the feasibility of this platform in questions. In order to address the complexity of a sample, this detection method can be further integrated with DNA extraction platforms, which can feed 19


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DNA samples into a nano/microchannel, such as a centrifugal microfluidic device for the extraction of pathogenic DNA from whole blood74, a microfluidic chip to isolate DNA in a single cell by steps from cell capture, cell lysis to DNA purification75, 76 and a fully-integrated biochip for the preparation of DNA from cells77. Especially for the concern about the concentration of background electrolytes in sample, it has been confirmed that ICP can occur under the ionic strength of physiological conditions. There have been several studies on the preconcentration of biomolecules using ICP phenomenon in 1X PBS. Kwak et al. demonstrated continuous-flow concentration with recombinant green fluorescence protein, red blood cells and Escherichia coli78. Liu et al. integrated ICP preconcentrator with a surface-based immunoassay for R-Phycoerythrin79. Kim et al. succeeded in fabricating a non-destructive micro/nanofluidic preconcentrator for red blood cells38. Even though the mobility of biomolecules varies, the critical mobility can be easily adjusted by modifying parameters in the equation. After all, physiological conditions for real samples can be handled by integrating microfluidic DNA extraction methods and by adjusting the critical mobility in this work. In addition, DNA molecules can be labeled with intercalating dyes such as YOYO and TOTO. It has been reported that RNA-guided dCas9 still has good affinity to dye-intercalated DNA44. Even though DNA molecules are labeled with intercalating dyes, target DNA would be distinguished as long as the critical mobility is set between the mobility of free DNA molecules and that of dCas9-DNA complex. All things considered, the proposed dCas9-mediated ICP platform would provide an effective and direct means for micro/nanofluidic liquid biopsy to diagnose disease-related DNA sequences.

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Supporting Information

The supporting Information is available free of charge on the ACS website at DOI: XXX.XXXXX Details for analytical derivation, numerical methods, additional simulation and experimental results, experimental setup and chemical preparations (pdf) Video1. Numerical and experimental verification of stacking and propagating behaviors (avi) Video2. Electrohydrodynamic preconcentration of free DNA, dCas9 and dCas9-DNA complex (avi)

Author contributions

H. Lee performed theoretical analysis and numerical simulation; J. Choi designed the micro/nanofluidic channel to conduct experiments for the concentrating behaviors; E. Jeong provided the dCas9 capturing system and conducted the gene detecting experiments with J. Choi; S. Baek conducted preliminary experiment with real DNA sample; H. C. Kim, J.-H. Chae, Y. Koh and S. W. Seo advised the medical insights; H. Lee, J. S. Kim and S. J. Kim supervised the project; all authors wrote the paper.

Competing financial interests

The authors declare no competing financial interests

Acknowledgements

H. Lee, J. Choi and S. J. Kim were supported by Basic Research Laboratory Project (NRF2018R1A4A1022513), Basic Science Research Program (2016R1A1A1A05005032 and 21


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2016R1A6A3A11930759), the Center for Integrated Smart Sensor (CISS- 2011-0031870) by the Ministry of Science and ICT and Korean Health Technology RND project (HI13C1468, HI14C0559). E. Jeong and J. S. Kim were supported by the Institute for Basic Science (IBSR021-D1). Also this work partially supported by BK21 plus program.

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Figure captions Figure 1. (a) Schematic diagram of voltage configurations and electrohydrodynamic force

balance on negatively charged analyte near a nanojunction. (b) An image for nanojunctionintegrated micro/nanofluidic device. (c) Conceptual images for identifying the presence of targeted DNA.

Figure 2. Numerical and experimental verifications of staking and propagating behaviors. The

Langevin dynamics simulations for (a) A = 0.9 cr and (b) A = 1.1 cr. Experimental verifications using (c) sulforhodamine B (A = 1.26 × 10-8 m2/V/s = 0.47 cr) and (d) Alexa488 (A = 3.61 × 10-8 m2/V/s = 1.34 cr).

Figure 3. The electrohydrodynamic behaviors of (a) free DNA, (b) dCas9 and (c) dCas9-DNA

complex. Their electrophoretic mobilities were calculated as 1.34 cr, 0.41 cr, 0.85 cr, respectively, where cr = 2.7 × 10-8 m2/V/s. The voltage configurations (VLOW = 15 V and VHIGH = 30 V) and the scale bar were the same in all experiments.

Figure 4. Off-target tests depending on the location of double-base mismatch between the

binding sequence and the guide RNA sequence. Fluorescence intensity was a function of distance from the nanojunction. The mismatches occurred at (a) 1, 2 positions, (b) 6, 7 positions, (c) 11, 12 positions and (d) 19, 20 positions far from the PAM. The applied voltage was 15 V for VLOW and 30 V for VHIGH.

Figure 5. Identification tests depending on DNA length for (a) relatively short DNA (50 bp and 23


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100 bp) and (b) relatively long DNA (750 bp and 1023 bp). The applied voltage was 15 V for VLOW and 30 V for VHIGH.

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References (1) International Human Genome Sequencing Consortium. Nature 2004, 431, (7011), 931-945. (2) Mitra, R. D.; Church, G. M. Nucleic Acids Research 1999, 27, (24), e34-e39. (3) Metzker, M. L. Nature reviews genetics 2010, 11, (1), 31-46. (4) Crowley, E.; Di Nicolantonio, F.; Loupakis, F.; Bardelli, A. Nature reviews Clinical oncology 2013, 10, (8), 472-484. (5) Bettegowda, C.; Sausen, M.; Leary, R. J.; Kinde, I.; Wang, Y.; Agrawal, N.; Bartlett, B. R.; Wang, H.; Luber, B.; Alani, R. M. Science translational medicine 2014, 6, (224), 224ra24-224ra24. (6) Cohen, A.; Terabe, S.; Smith, J. A.; Karger, B. Analytical chemistry 1987, 59, (7), 1021-1027. (7) Grossman, P. D., Free-solution capillary electrophoresis. Academic Press: San Diego: 1992. (8) Stellwagen, N. C.; Gelfi, C.; Righetti, P. G. Biopolymers 1997, 42, (6), 687-703. (9) Viovy, J.-L. Reviews of Modern Physics 2000, 72, (3), 813. (10) Meagher, R. J.; Won, J. I.; McCormick, L. C.; Nedelcu, S.; Bertrand, M. M.; Bertram, J. L.; Drouin, G.; Barron, A. E.; Slater, G. W. Electrophoresis 2005, 26, (2), 331-350. (11) Shagin, D. A.; Shagina, I. A.; Zaretsky, A. R.; Barsova, E. V.; Kelmanson, I. V.; Lukyanov, S.; Chudakov, D. M.; Shugay, M. Scientific Reports 2017, 7. (12) Clarke, L.; Rebelo, C.; Goncalves, J.; Boavida, M.; Jordan, P. Molecular Pathology 2001, 54, (5), 351. (13) Kwak, D.-K.; Chae, H.; Lee, M.-K.; Ha, J.-H.; Goyal, G.; Kim, M. J.; Kim, K.-B.; Chi, S.-W. Angewandte Chemie International Edition 2016, 55, (19), 5713-5717. (14) Lee, K.; Lee, H.; Lee, S.-H.; Kim, H.-M.; Kim, K.-B.; Kim, S. J. Nanoscale 2017, 9, (45), 18012-18021. (15) Kim, D.-S.; Jeong, Y.-T.; Park, H.-J.; Shin, J.-K.; Choi, P.; Lee, J.-H.; Lim, G. Biosensors and Bioelectronics 2004, 20, (1), 69-74. (16) Lee, S.-H.; Lee, H.; Jin, T.; Park, S.; Yoon, B. J.; Sung, G. Y.; Kim, K.-B.; Kim, S. J. Nanoscale 2015, 7, (3), 936-946. (17) Mani, A.; Zangle, T. A.; Santiago, J. G. Langmuir 2009, 25, (6), 3898-3908. (18) Schoch, R. B.; Han, J.; Renaud, P. Rev. Mod. Phys. 2008, 80, 839-883. (19) Kim, S. J.; Li, L. D.; Han, J. Langmuir 2009, 25, (13), 7759-7765. (20) Kim, S. J.; Ko, S. H.; Kwak, R.; Posner, J. D.; Kang, K. H.; Han, J. Nanoscale 2012, 4, (23), 7406-7410. (21) Kim, S. J.; Ko, S. H.; Kang, K. H.; Han, J. Nat Nano 2010, 5, (4), 297-301. (22) Kim, W.; Park, S.; Kim, K.; Kim, S. J. Lab Chip 2017, 17, (22), 3841-3850. (23) Jin, X.; Joseph, S.; Gatimu, E. N.; Bohn, P. W.; Aluru, N. R. Langmuir 2007, 23, 13209-13222. (24) Rubinstein, I.; Shtilman, L. Journal of the Chemical Society, Faraday Transactions 2: Molecular and Chemical Physics 1979, 75, (0), 231-246. (25) Demekhin, E. A.; Nikitin, N. V.; Shelistov, V. S. Physics of Fluids 2013, 25, (12). (26) Zaltzman, B.; Rubinstein, I. J. Fluid Mech. 2007, 579, 173-226. (27) Cho, I.; Kim, W.; Kim, J.; Kim, H.-Y.; Lee, H.; Kim, S. J. Phys. Rev. Lett. 2016, 116, (25), 254501. (28) Rubinstein, I.; Zaltzman, B. Physical Review E 2000, 62, (2), 2238-2251. (29) Rubinstein, S. M.; Manukyan, G.; Staicu, A.; Rubinstein, I.; Zaltzman, B.; Lammertink, R. G. H.; Mugele, F.; Wessling, M. Phys. Rev. Lett. 2008, 101, 236101. (30) Kim, S. J.; Wang, Y.-C.; Lee, J. H.; Jang, H.; Han, J. Phys. Rev. Lett. 2007, 99, 044501. (31) Kim, S. J.; Song, Y.-A.; Han, J. Chemical Society Reviews 2010, 39, (3), 912-922. (32) Dydek, E. V.; Zaltzman, B.; Rubinstein, I.; Deng, D. S.; Mani, A.; Bazant, M. Z. Phys. Rev. Lett. 2011, 107, 118301. (33) Sohn, S.; Cho, I.; Kwon, S.; Lee, H.; Kim, S. J. Langmuir 2018, 34, (26), 7916-7921. (34) Martins, D.; Wei, X.; Levicky, R.; Song, Y.-A. Analytical Chemistry 2016, 88, (7), 3539-3547. (35) Hong, S. A.; Kim, Y.-J.; Kim, S. J.; Yang, S. Biosensors and Bioelectronics 2018, 107, 103-110. (36) Ko, S. H.; Kim, S. J.; Cheow, L. F.; Li, L. D.; Kang, K. H.; Han, J. Lab on a Chip 2011, 11, (7), 13511358. (37) Lee, J. H.; Cosgrove, B. D.; Lauffenburger, D. A.; Han, J. J. Am. Chem. Soc. 2009, 131, (30), 1034010341. (38) Kim, J.; Cho, I.; Lee, H.; Kim, S. J. Sci Rep 2017, 7, (1), 5091. (39) Plecis, A.; Nanteuil, C.; Haghiri-Gosnet, A.-M.; Chen, Y. Anal. Chem. 2008, 80, (24), 9542-9550. (40) Cheow, L. F.; Sarkar, A.; Kolitz, S.; Lauffenburger, D.; Han, J. Anal. Chem. 2014, 86, (15), 7455-7462. (41) Cheow, L. F.; Han, J. Y. Anal. Chem. 2011, 83, (18), 7086-7093. (42) Choi, J.; Huh, K.; Moon, D. J.; Lee, H.; Son, S. Y.; Kim, K.; Kim, H. C.; Chae, J.-H.; Sung, G. Y.; Kim, 25


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H.-Y.; Hong, J. W.; Kim, S. J. RSC Advances 2015, 5, (81), 66178-66184. (43) Wu, X.; Scott, D. A.; Kriz, A. J.; Chiu, A. C.; Hsu, P. D.; Dadon, D. B.; Cheng, A. W.; Trevino, A. E.; Konermann, S.; Chen, S. Nature biotechnology 2014, 32, (7), 670-676. (44) Sternberg, S. H.; Redding, S.; Jinek, M.; Greene, E. C.; Doudna, J. A. Nature 2014, 507, (7490), 62-67. (45) Abudayyeh, O. O.; Gootenberg, J. S.; Konermann, S.; Joung, J.; Slaymaker, I. M.; Cox, D. B.; Shmakov, S.; Makarova, K. S.; Semenova, E.; Minakhin, L. Science 2016, 353, (6299), aaf5573. (46) Lee, S. J.; Rhee, H.; Jeon, T.-J.; Kim, D. Sensors and Actuators B: Chemical 2016, 229, (Supplement C), 276-280. (47) Son, S. Y.; Lee, S.; Lee, H.; Kim, S. J. BioChip Journal 2016, 10, (4), 251-261. (48) Nam, S.; Cho, I.; Heo, J.; Lim, G.; Bazant, M. Z.; Moon, D. J.; Sung, G. Y.; Kim, S. J. Phys. Rev. Lett. 2015, 114, (11), 114501. (49) Alizadeh, S.; Mani, A. Langmuir 2017, 33, (25), 6205-6219. (50) Ko, S. H.; Song, Y.-A.; Kim, S. J.; Kim, M.; Han, J.; Kang, K. H. Lab on a Chip 2012, 12, (21), 44724482. (51) Yossifon, G.; Chang, H.-C. Phys. Rev. Lett. 2008, 101, 254501. (52) Masliyah, J. H.; Bhattacharjee, S., Electrokinetic and Colloid Transport Phenomena. Wiley: 2006. (53) Merhari, L., Hybrid Nanocomposites for Nanotechnology: Electronic, Optical, Magnetic and Biomedical Applications. Springer US: 2009. (54) Milanova, D.; Chambers, R. D.; Bahga, S. S.; Santiago, J. G. Electrophoresis 2011, 32, (22), 3286-3294. (55) Gong, L.; Ouyang, W.; Li, Z.; Han, J. AIP advances 2017, 7, (12), 125020. (56) Jinek, M.; Jiang, F.; Taylor, D. W.; Sternberg, S. H.; Kaya, E.; Ma, E.; Anders, C.; Hauer, M.; Zhou, K.; Lin, S. Science 2014, 343, (6176), 1247997. (57) Shibata, M.; Nishimasu, H.; Kodera, N.; Hirano, S.; Ando, T.; Uchihashi, T.; Nureki, O. Nature communications 2017, 8, (1), 1430. (58) Sun, W.; Ji, W.; Hall, J. M.; Hu, Q.; Wang, C.; Beisel, C. L.; Gu, Z. Angewandte Chemie International Edition 2015, 54, (41), 12029-12033. (59) Keyser, U. F.; Koeleman, B. N.; Van Dorp, S.; Krapf, D.; Smeets, R. M.; Lemay, S. G.; Dekker, N. H.; Dekker, C. Nature Physics 2006, 2, (7), 473-477. (60) Teraoka, I. Progress in Polymer Science 1996, 21, (1), 89-149. (61) Li, M.; Wang, I. X.; Li, Y.; Bruzel, A.; Richards, A. L.; Toung, J. M.; Cheung, V. G. Science 2011, 1207018. (62) Naeim, F., Atlas of hematopathology: morphology, immunophenotype, cytogenetics, and molecular approaches. Academic Press: 2012. (63) Moscou, M. J.; Bogdanove, A. J. Science 2009, 326, (5959), 1501-1501. (64) Tuerk, C.; Gold, L. Science 1990, 249, 505-510. (65) Pavletich, N. P.; Pabo, C. O. Science 1991, 252, (5007), 809-817. (66) Jinek, M.; Chylinski, K.; Fonfara, I.; Hauer, M.; Doudna, J. A.; Charpentier, E. Science 2012, 337, (6096), 816-821. (67) Qi, L. S.; Larson, M. H.; Gilbert, L. A.; Doudna, J. A.; Weissman, J. S.; Arkin, A. P.; Lim, W. A. Cell 2013, 152, (5), 1173-1183. (68) Anders, C.; Niewoehner, O.; Duerst, A.; Jinek, M. Nature 2014, 513, (7519), 569-573. (69) Liu, L.; Yu, T.; Wang, L.; Mo, X.; Yu, Y. Meta gene 2014, 2, 469-478. (70) McInnes, B.; Brown, C. A.; Mahuran, D. J. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 1992, 1138, (4), 315-317. (71) Zollino, M.; Orteschi, D.; Murdolo, M.; Lattante, S.; Battaglia, D.; Stefanini, C.; Mercuri, E.; Chiurazzi, P.; Neri, G.; Marangi, G. Nature genetics 2012, 44, (6), 636. (72) Chen, Y.; Jia, X.; Wang, P.; Xiao, X.; Li, S.; Guo, X.; Zhang, Q. Molecular vision 2013, 19, 292. (73) Fan, H. C.; Blumenfeld, Y. J.; Chitkara, U.; Hudgins, L.; Quake, S. R. Clinical chemistry 2010, 56, (8), 1279-1286. (74) Cho, Y.-K.; Lee, J.-G.; Park, J.-M.; Lee, B.-S.; Lee, Y.; Ko, C. Lab Chip 2007, 7, (5), 565-573. (75) Marcus, J. S.; Anderson, W. F.; Quake, S. R. Anal. Chem. 2006, 78, (9), 3084-3089. (76) Kim, J.; Johnson, M.; Hill, P.; Gale, B. K. Integrative Biology 2009, 1, (10), 574-586. (77) Liu, R. H.; Yang, J.; Lenigk, R.; Bonanno, J.; Grodzinski, P. Anal. Chem. 2004, 76, (7), 1824-1831. (78) Kwak, R.; Kim, S. J.; Han, J. Anal. Chem. 2011, 83, 7348-7355. (79) Liu, V.; Song, Y.-A.; Han, J. Lab Chip 2010, 10, 1485-1490. 26


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(a)

mismatch PAM 3’ (b) 3’ 3’ 3’ 5’ 5’ 5’ 5’ 18–2– PAM 13–2–5- PAM 250 m

5 min 8 min 15 min

Fluorescence intensity (a.u.)

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Distance from nanojunction (m)

(c) 3’

5’ 8–2–10- PAM

3’ 5’

(d) 3’ 5’

30 Figure 4


2–18- PAM

3’ 5’

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(a)

50 bp

100 bp

200 m 3.5 min

3.5 min

6.5 min

6.5 min

7.5 min

7.5 min

10 min

10 min

complex  cr   DNA (stacking) 750 bp

(propagating) 1023 bp

(b) 3.5 min

2 min

6.5 min

3 min

7.5 min

4 min

10 min

5 min

cr  complex   DNA

(propagating) (propagating)

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dCas9-mediated Nanoelectrokinetic Direct Detection of Target Gene

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