Fm to Am Nucleic Acid Amplification for Molecular Diagnostics in a Silicone-Coated Metal

1SUPPORTING INFORMATIONS

3 fM to aM nucleic acid amplification for molecular diagnostics in a

4 silicone-coated metal microfluidic bioreactor

6 Guoliang Huang 1,2, Qin Huang 2, Li Ma2, Xianbo Luo2, Biao Pang2, Zhixin Zhang2, Yuliang Wang1, 7 Junqi Zhang1, Qi Li1, Rongxin Fu1, and Jiancheng Ye1

9 1Department of Biomedical Engineering, the School of Medicine, Tsinghua University, Beijing

10 100084, China.

11 2National Engineering Research Center for Beijing Biochip Technology, Beijing 102206, China.

12 Correspondence and requests for materials should be addressed to G.L.H. ([email protected])

1 1 1Supporting Information

2Results

3Comparison of isothermal DNA amplification in uncoated and non-stick-coated metal micro-

4nanoliter fluidic chips. Using the same DNA template concentration of 1.3 fM (10-15 M) and 7-μ L

5reaction mixtures in every bioreactor cell, isothermal DNA amplification in uncoated and non-stick

6coating metal micro-nanoliter fluidic chips was performed using our portable confocal detector. The

7contrast of DNA isothermal amplification curves is shown in Figure S1. Figure S1 (a) corresponds to

8isothermal DNA amplification in the uncoated metal micro-nanoliter fluidic chip, where the times at

9the second derivative inflexions of the exponential DNA amplification curves for the five bioreactor

10cells were 24.85, 27.29, 27.80, 28.18, and 29.53 min. The maximum of the time difference at these

11inflexions was ~4.68 min. At the top of the amplification curves, the fluorescence intensities of the

12five bioreactor cells ranged from 3600-6900; the percent deviation of the fluorescence intensity was

13~91.7%. Figure S1 (b) is isothermal DNA amplification in the non-stick-coated metal micro-

14nanoliter fluidic chip, where the times at the second derivative inflexions of the exponential DNA

15amplification curves for the five bioreactor cells corresponded to 19.42, 19.45, 19.59, 19.60, and

1619.92 min. The maximum of the time differences at these inflexions was 0.5 min. At the top of the

17amplification curves, the fluorescence intensities of the five bioreactor cells ranged from 4000-4500;

18the percent deviation of the fluorescence intensity was ~11%. Figure S1 (c) is the normalizing

19processing of the isothermal DNA amplification curves in Figure S1 (b), which is generally used in

20commercial RT-PCR setups.

1 2 (a)

1 3 1 2 Figure S1 3 4 Table S1

5 DNA template (fM) 1.3×102 1.3×101 1.3×100 1.3×10-1 1.3×10-2 1.3×10-3

Time at the inflexion (min) In 7-μL non-stick-coated metal micro-nanoliter fluidic bioreactors measured using our 14.8 17.4 19.6 21.5 24.5 28.7 Portable Confocal Detection System In 25-μL Eppendorf tubes non measured using an ABI 7700 16.3 18.4 20.8 23.7 28.9 amplifi Fast Real-Time PCR System cation 6

7DISCUSSION

8 The fluorescent detection response of nucleic acid amplification is related to many factors,

9including the number of amplified double-stranded DNAs (dsDNAs), the number of denatured

10dsDNAs at the amplification temperature, the surface adsorption of the bioreactor, photo-bleaching,

11and background noise. The continuously increasing number of amplified dsDNAs produces a

12positive or upper dynamical amplified signal. However, the denatured dsDNA at 65°C, the surface

13adsorption of DNA by the bioreactor, and photo-bleaching from the excitation light lead to a

14negative or downward dynamical change of the amplified signal, which usually causes the detection

15response of the nucleic acid amplification to be unstable and late1,2. Further, the background noise of

16the bioreactor material and surroundings produces a high original value for the dynamical amplified

17signal and low amplified efficiency or low ratio of end signal to start signal, which usually decreases

18sensitivity. Therefore, general nucleic acid amplification reactions in Eppendorf tubes and 96- or

19384-well microplates is usually performed in ≥25-μL volumes to increase the signal of the

1 4 1amplified dsDNA far more than the lost signal from the negative factors, as well as to obtain a

2relative steady exponential mode for the amplified signal and good consistency for repeated

3detection. Indeed, in these conditions, the differences from the denatured dsDNA at the high

4amplification temperature, the surface adsorption of the bioreactor, photo-bleaching, and background

5noise can be ignored.

6 When the reaction volume is reduced to <10 μL, the above negative effects are significant. If the

7lost fluorescence signal from the denatured dsDNA is Eu, the increasing fluorescence signal from the

8amplified dsDNA is Ea, the decreased fluorescence signal from surface adsorption is Es, the reduced

9fluorescence signal from photo-bleaching is Ep, and the background signal from the bioreactor

10material and surroundings is Eb, then the dynamic measured fluorescence signal of nucleic acid

11amplification E can be calculated with the following formula (1):

12 E = Eo + Ea + Eb - Es - Eu - Ep (1)

13where Eo is the original fluorescence signal of the mixtures, including the template DNA, sample,

14and reagents at 25°C. We report that the non-stick-coated metal micro-nanoliter fluidic chip and

15portable confocal detector decreased these possible negative effects, resulting in stable nucleic acid

16amplification, sensitive detection, and a rapid response in micro-nanoliter reaction volumes.

17 Figure S1 shows an obvious change in the nucleic acid amplification from the surface adsorption

18in uncoated and non-stick-coated metal micro-nanoliter fluidic bioreactors. In Figure S1 (a), the

19uncoated metal micro-nanoliter fluidic chip displays a larger difference both in the times at the

20second derivative inflexion of the exponential DNA amplification curves (~4.7 min) and the relative

21fluorescence intensity (~91.7%). In Figure S1 (b), when the metal micro-nanoliter fluidic bioreactor

22was coated with the silicone non-stick material, the surface of the bioreactor became smooth and

23inert to DNA molecules, effectively eliminating surface adsorption (Es), which makes the nucleic

24acid amplification stable with little difference in the times at the second derivative inflexion of the

25exponential DNA amplification curves (~0.5 min) and the relative fluorescence intensity (~11%).

1 5 1 Figure S2 displays our portable confocal detector, which can collect fluorescence near the optical

2diffraction limit and effectively limit the background noise (Fig. S5). The fluorescence from an

3object on the focal plane can be focused into a small spot and transmitted with >92% intensity to the

4detector behind a pinhole filter (PH) at the focal plane of the imaging lens set (L2). The fluorescence

5from other off-focus objects creates a large dispersive spot with a very low transmitted efficiency

6behind the PH (e.g., the transmitted efficiency at the off-focus position of 18 μm is only ~1.13%).

7The detection plane for the fluorescence of the bioreactor material and surrounding is farther than

8the off-focus position of 50 μm, and thus, the background noise (Eb) from the bioreactor material

9and surroundings is approximately 0 and can be ignored.

-t/T 10 The photo-bleaching from the excitation light is directly proportional to 1-e , where T is half of

11the attenuant period and t is the irradiation time with the excitation light3. In our portable confocal

12detector, the micro-nanoliter fluidic bioreactor was moved by a rotary scanning stage (RS), and

13every bioreactor cell in one measuring period of 30 s was irradiated for <10 ms. Therefore, the

14photo-bleaching from the excitation light (Ep) was approximately 0. High amplification efficiency

15and a negligible effect from denatured dsDNA can be obtained by optimizing the amplification

16conditions, including the temperature (65°C) and the addition of 0.5 mg/ml BSA and 6 mM MgS04,

17which renders the lost fluorescence signal from denatured dsDNA (Eu) stable as a constant.

18 After all of the effects from the surface adsorption of the bioreactor, the denatured dsDNA at the

19amplification temperature, the photo-bleaching from the excitation light, and the background from

20the bioreactor material and surroundings are limited, and equation (1) can is simplified as:

21 E = Ea + C (2)

22where C is a constant, C = Eo - Eu. Compared to equation (1), equation (2) indicates that the

23dynamic fluorescence signal E of the nucleic acid amplification is directly proportional to the

24increased fluorescence signal Ea of amplified dsDNAs and can immediately respond to amplified

25dsDNAs in real time. Thus, the sensitivity can be improved when the negative factors are eliminated.

2 3 4 Figure S2

6Methods

7Design of primers. Oligonucleotide primers were designed for the isothermal amplified assay according to the sequence of the

8Mycoplasma pneumoniae P1 gene from GenBank (Accession No. M18639). Six primers, including two loop primers (LF and

9LB), two outer primers (F3 and B3), and two inner primers (FIP and BIP), were designed to recognize eight distinct regions on

10the target sequence (Fig. S3). BIP consists of the complementary sequence of B1 and the sense sequence of B2, FIP is

11comprised of the complementary sequence of F1 and the sense sequence of F2. The primer sequences are listed in Figure S4.

1 7 1 2 3 Figure S3 4

5 6 7 Figure S4 8

9Portable real-time fluorescent confocal detector. To detect the fluorescent signal of DNA amplification in real time with

10high sensitivity and low background, a new portable confocal detector was developed as shown in Figure S5. In Figure S5, an

11objective set L1 with a focal length of 13 mm and numerical aperture of NA = 0.72 was designed, which consists of seven

12lenses, including two doublets, and uses only three glass materials, ZK7, ZF2, and ZK11. The excited light from a white LED

13was first collimated by an aspherical lens L1 and filtered by the band pass filter F1 with a central wavelength of 480 nm and

14bandwidth of 30 nm. Then, the excited light was focused by the objective set L2 to illuminate the bioreactor cells of the micro-

15nanoliter fluidic chip MC on the rotary scanning stage RS. The intensity of the excited light was adjusted to adapt to the

16different applications of the chip by the attenuator A1. A dichroic mirror D1 was used to reflect the fluorescence from the

17bioreactor cell and to penetrate the excited light from the LED. The fluorescence from Sybase Green inserting into the dsDNA

18in the bioreactor cell was excited by the LED and initially collected by the objective set L2, reflected by D1, and focused onto

1 8 1the detector PMT (HAMAMATSU, Japan) by the imaging lens set L3 with a focal length of 22 mm. A filter F2 with a 520-nm

2central wavelength and 40-nm bandwidth was used to penetrate the fluorescence and limit the excited light. The pinhole PH

3was used to filter the farraginous light from the environment and the off-focused fluorescence from the material of the micro-

4nanoliter fluidic chip. Finally, the fluorescence signal was transferred to a computer by an A/D processor. The temperature

5controller TCD was used to administer the heater HF with an accuracy of 0.1°C and speed of raising the temperature of 1°C/s

6by the temperature feedback of sensor S in the process of DNA isothermal amplification. A multiple-axis moving driver

7MAMD was used to supervise the rotary scanning stage RS with angle accuracy of 0.01°.

8When the micro-nanoliter fluidic chip was used, the inlet and outlet holes were first opened using an Eppendorf tip with light

9pressure. Then, the mixtures of the circular probes, reagents, and DNA samples were injected into the bioreactor cells of the

10micro-nanoliter fluidic chip from the inlet hole using the tip. Third, the inlet hole and the outlet hole were sealed with a thin PC

11film from ABI Corporation. Fourth, the thin PC film, exceeding the edge of the chip or covering the center fixed hole of the

12chip, was trimmed for detection. Fifth, the channel between any two adjacent bioreactor cells was obstructed by heating

13compression to keep all bioreactor cells independent of each other during the process of isothermal amplification. Finally, the

14micro-nanoliter fluidic chip was placed into the portable confocal detector (Fig. S5) for isothermal DNA amplification and

15gene-specific identification.

Figure S5 17

1 9 1References

21. Wang, M. et al. Accelerated Photobleaching of a Cyanine Dye in the Presence of a Ternary Target DNA, PNA

3 Probe, Dye Catalytic Complex: A Molecular Diagnostic. Anal. Chem. 81, 2043–2052 (2009).

42. Koek, M.M. et al. Metabolic Profiling of Ultrasmall Sample Volumes with GC/MS: From Microliter to Nanoliter

5 Samples. Anal. Chem. 82, 156–162 (2010).

63. Huang, G.L. et al. Fluorescence photobleaching properties for microarray chips. Chinese Journal of Luminescence.

7 Chinese Journal of Luminescence 27, 259-264 (2006). (in Chinese)

1 10 1Figure caption

2Figure S1 Comparison of DNA amplification in uncoated and non-stick-coated metal micro-

3nanoliter fluidic chips. (a) Isothermal DNA amplification in the uncoated metal micro-nanoliter

4fluidic chip, which displays obvious differences among the five parallel bioreactors. (b) Isothermal

5DNA amplification in the non-stick-coated metal micro-nanoliter fluidic chip, where the

6amplification in the five parallel bioreactors displays good consistency and the time difference at the

7second derivative inflexions of the exponential DNA amplification curves for the five bioreactors are

8within 0.5 min. (c) Normalizing processing of the isothermal DNA amplification curves in (b).

10Table S1 Comparison of the times at the inflexions of the second derivative of the exponential

11DNA amplification curves between our advanced metal micro-nanoliter fluidic bioreactors and

12in the general 25-μL Eppendorf tubes.

14Figure S2 Comparison of fluorescence collection on the focal plane to the off-focus position for

15the developed portable confocal detector. The horizontal axis corresponds to the radius of the

16optical spot from the center of the optical axis, and the vertical axis is the relative transmitted

17intensity. The fluorescent intensity on the focal plane can be collected with a high efficiency (92%)

18near the optical diffractive limit. There was a low transmitted efficiency of 3.46% at the off-focus

19position of 8 μm and lower transmitted efficiency of 1.13% at the off focus position of 18 μm.

21Figure S3 Diagram of the primers used. Constructions of the inner primers BIP and FIP are

22displayed. F1c and B1c are complementary sequences to F1 and B1, respectively.

1 11 1Figure S4 Partial nucleotide sequences of the P1 gene of M. pneumoniae and the primers used

2for amplification. Arrows indicate positions of specific primers. The left arrow indicates that a

3complementary sequence is used for the primer. The right arrow indicates that a sense sequence is

4used for the primer.

5Figure S5 Portable confocal detector. The white light LED had a power of 120 mW, aspherical

6lens L1 had a focal length of 20 mm, A1 is an attenuator, F1 and F2 are two band pass filters, D1 is a

7dichroic mirror, and L2 is an objective. MC is the microfluidic chip, S is the temperature sensor, RS

8is the rotary scanning stage, HF is the heater, L3 is an imaging lens set, PH is the pinhole, and PMT

9is a photo-electronic detector. A/D is a 16-bit analog-to-digital processor, TCD is the temperature

10controller, MAMD is a multiple-axis moving driver, and a computer was used to manage the process

11of isothermal DNA amplification and display the amplification curves in real time.