Inkjet-printed fluidic paper devices for chemical and biological ... - Eric Hoppmann · 2019. 7....

Author’s Accepted Manuscript JSTQE (2014) http://dx.doi.org/10.1109/JSTQE.2013.2286076 © 2014 IEEE. Personal use of this material is permitted. Permission from IEEE must be obtained for all uses.

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Abstract—The fabrication of substrates for surface enhanced

Raman spectroscopy (SERS) by printing plasmonic structures on paper has emerged as a potential low-cost replacement for conventional nanofabricated SERS devices. Not only are the paper devices low in cost to produce, the inherent flexibility and fluidic transport capabilities of paper provide advantages in sample collection and processing, as well as analyte concentration. In this work, we review the recent progress in paper or membrane SERS devices fabricated through inkjet printing and other deposition methods. We then report a new potential diagnostics application for paper SERS devices that leverages the fluidic transport and chromatographic capabilities of paper to enable SERS-based detection following PCR. The use of paper SERS creates the potential for a simple yet densely multiplexed PCR assay that is not possible with conventional fluorescence-based transduction.

Index Terms—Paper, Surface enhanced Raman spectroscopy, SERS, chemical sensing, biosensing, polymerase chain reaction. PCR, TaqMan.

I. INTRODUCTION URFACE enhanced Raman spectroscopy (SERS) boasts the capability for label-free chemical analysis with the

detection performance of fluorescence spectroscopy [1] and a multiplexing density that surpasses that of fluorescence techniques [2], [3]. Upon laser light excitation, Raman-scattered photons from a molecule reveal the landscape of vibrational energy states, which is unique to the molecule. Thus, detection of the Raman scattered photons provides a spectral fingerprint that can be used to identify the molecule and its characteristics. Unfortunately, Raman scattering is an extremely weak effect and thus it cannot generally be applied to the detection of trace quantities of analytes in its conventional form. Nearly forty years ago, however, it was discovered that noble metal nanostructures provide a boost of many orders of magnitude to the Raman signal for molecules interacting at the surface [4–7]. This effect, surface enhanced Raman scattering, is the result of the combination of an electromagnetic enhancement provided by the localized

Manuscript received July 31, 2013. This work was supported by the National Science Foundation (ECCS1149850) and the University of Maryland Center of Excellence in Regulatory Science and Innovation (CERSI).

E. P. Hoppmann, W. W. Yu, and I. M. White are with the Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, USA ([email protected], 301.405.6230). E. P. Hoppmann and W. W. Yu contributed equally to this work.

surface plasmon resonances at the metal nanostructure surface [8], [9] as well as a chemical effect at the metal surface [10]. The powerful performance capability of the SERS technique was demonstrated more than 15 years ago with the reports of single molecule identification using SERS [1], [10–12].

Despite these successes in the research lab, currently SERS is not widely utilized for real world applications in chemical analysis or clinical diagnostics. Instead, a host of other analytical technologies, especially chromatography with mass spectrometry (MS), are used for these mainstream applications. These techniques are well established for laboratory use and have excellent performance; thus, it is unlikely that SERS will displace current lab-based analytical techniques based on performance. However, given the high cost and bulky nature of chromatography + MS, pairing low cost SERS devices with newly available portable Raman spectrometers can enable SERS to break into real world applications by offering a dramatic decrease in the cost of analysis, and by providing portability for routine analysis in the field.

Today, the most common method for performing SERS measurements is to deposit a droplet of a liquid sample onto a rigid silicon or glass substrate that has a nanostructured noble metal surface. When the sample dries, analyte molecules within the sample adsorb onto the nanostructured metal surface, where they will experience the plasmonic and chemical enhancement associated with SERS. These SERS-active surfaces can be fabricated through a number of possible techniques, including self assembly [13–16], directed or templated assembly [17–20], thin film growth [21], and nanolithography [22–24]. While nanolithography approaches tend to have high SERS enhancement factors and superior uniformity, they are complex and expensive to produce, and they suffer from low throughput. Growth and assembly approaches are less expensive, but they do not offer much improvement in throughput, and they still require relatively sophisticated fabrication facilities. Hence, unfortunately current SERS devices are not yet optimized to meet the needs of routine detection of chemical species in the field.

II. PLASMONIC PAPER – A NEW LOW-COST SERS PARADIGM For SERS to reach low-cost applications in chemical and

biomolecule analytics – especially field-based applications – a significant shift in the methods for creating SERS devices is

Inkjet-printed fluidic paper devices for chemical and biological analytics using surface enhanced Raman spectroscopy

Eric P. Hoppmann, Wei W. Yu, and Ian M. White

S


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necessary. Recently, a number of reports have demonstrated the fabrication of plasmonic devices using low cost assembly (outside of a clean room) with paper or other flexible membranes as the substrate supports. To our knowledge, the very first reports of SERS utilizing paper date back to almost thirty years ago by Tran et al. [25], [26] However, in their experiments, paper was used to separate analytes by chromatography, after which silver colloid was then deposited for SERS measurements. Vo-Dinh, et al., reported the first use of plasmonic nanoparticles deposited on paper as SERS substrates [27], [28]. In this early work, Teflon or polystyrene latex spheres were deposited uniformly onto filter paper, followed by vacuum deposition of silver onto the spheres. These substrates were then used to detect various trace organic compounds. Following this, Berthod et al., [29–31] reported a different approach to fabricate paper SERS substrates. In their work, silver nanoparticles were grown in situ on the paper surface by soaking the paper in silver nitrate solution and then reducing the silver ions with sodium borohydride. A decade later, Feng, et al., reported the creation of plasmonic paper through drop-wise deposition of metal nanoparticles onto filter paper [32–34].

In the current decade, plasmonic paper has gained significant momentum as a possible replacement for the more expensive nanofabricated or microfabricated SERS substrates through novel approaches such as soaking, screen printing and inkjet printing. Lee, et al. fabricated plasmonic paper by soaking filter paper in colloid solution [35]. Additional reports of paper SERS devices from the same group (Singamaneni, et al.) followed [36], [37], including the detection of 500 pM trans-1,2-Bis(4-pyridyl)ethylene (BPE), a standard SERS model analyte. Ngo, et al., further studied the system of plasmonic paper fabricated through soaking, reporting techniques to control surface coverage and aggregation of metal nanostructures on paper [38], [39]. An additional study on soaking-based fabrication was reported by Mehn, et al., in which a method for the controlled deposition of metal nanostars was developed [40]. In addition to this series of reports on direct deposition of nanoparticles onto paper through soaking, Cheng, et al., returned to the concept of in-situ synthesis with a detailed study of the synthesis parameters for plasmonic structures in paper [41].

Our group explored inkjet printing as a simple and inexpensive approach for the fabrication of plasmonic paper devices [42]. Inkjet printing has previously been utilized for conductive circuits [43–45], as well as SERS active patterns using nanoparticle inks [46]. In our approach, nanoparticle ink was formed by adding appropriate amounts of glycerol and ethanol to a highly concentrated silver colloid. The ink was then loaded into replacement cartridges in a low-cost consumer desktop inkjet printer and printed onto filter paper. Additionally, the paper was hydrophobically modified by inkjet printing hexadecenyl succinic anhydride (ASA) such that the aqueous sample would not spread when deposited onto the paper. Using this technique, we were able to detect as low as 10 femtomoles (~5 pg) of Rhodamine 6G (R6G), a standard SERS model analyte. We further analyzed the

performance of the inkjet-fabricated substrates by detecting BPE [47]. In this case, measurements of Raman intensity were obtained over a wide range of concentrations and fit to a Langmuir isotherm; as shown in Fig. 1, the data is an excellent fit (with low variation across substrates), suggesting that the inkjet-printed substrates may enable quantitative detection. The detection limit for BPE in this measurement is 20 femtomoles (~4 pg). Importantly, this analysis (and all subsequent chemical analysis by our group reviewed here) was performed with a relatively low cost and portable Raman system consisting of a 785 nm laser (Ocean Optics), fiber optic Raman probe (InPhotonics) and a QE65000 spectrometer (Ocean Optics).

Other groups have also demonstrated successful fabrication of plasmonic substrates using printing techniques. Fierro-Mercado, et al., reported the detection of explosives using inkjet-printed paper SERS substrates [48]. The detection of heavy metals using plasmonic metal nanoparticles that were inkjet-printed onto silicon substrates has also been reported [49]. As an alternative to inkjet-printing, Qu, et al., explored screen printing for SERS device fabrication. An excellent detection performance (160 fM R6G) was reported for screen-printed nanostructures on glass fiber [50].

Each of the fabrication approaches mentioned previously has its advantages and disadvantages. Techniques such as soaking, drop-wise deposition and in situ synthesis are extraordinarily simple, and do not require any equipment for SERS substrate fabrication. However, they also tend to be time consuming and are not conducive to large-scale production. Inkjet printing and screen printing are suitable for batch production, but these require additional machinery. In addition, nanoparticles need to be formulated into a suitable ink in order to be dispensed by the printers. Regardless of the approach taken to deposit nanoparticles onto the paper surface, this class of plasmonic substrates clearly offers a much lower-cost and higher-throughput methodology for fabrication of SERS devices. Importantly, despite the simplicity and low cost of fabrication, these devices have demonstrated excellent detection limits due to the high density of resonant plasmonic nanostructures embedded into the paper (scanning electron micrographs of a number of the substrates are presented in Fig. 2).

Fig. 1. BPE SERS signal intensity vs. concentration. Signal intensity is measured at 1207cm-1. Data is fitted using the Langmuir isotherm. Reprinted from Hoppmann, et al., [47], copyright 2013, Elsevier.


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A feature common to many these substrates is the random aggregation of the nanostructures on the paper surface, forming ‘hotspots’ which are responsible for the high SERS enhancement. With this randomness, one may expect a higher variability to the SERS signal from paper SERS substrates compared to nanofabricated periodic substrates. In practice, paper SERS substrates fabricated using the different deposition approaches have fairly low variability, typically in the range of 5 – 15% (see Table 1). This is because a typical Raman detection system will have a laser beam size which is much larger compared to the size of the nanostructures, hence the variability in the SERS enhancement are averaged out over

thousands of nanoparticle aggregates. For a more direct comparison with conventional plasmonic

substrates that are fabricated with nanofabrication, we can consider the enhancement factor, which is generally defined as the factor of improvement in Raman signal with SERS as compared to without SERS, given a normalized optical system and number of analyte molecules within the detection volume. The low-cost substrates discussed above typically have an enhancement factor on the order of 105 to 108 (see Table 1). By comparison, nanofabricated substrates tend to have an enhancement factor near 108. Thus, in a direct comparison, one should expect somewhat improved detection performance by utilizing the expensive nanofabricated substrates; however, the tremendous cost advantage of these new paper substrates, combined with the strong detection performance, may be sufficient for many applications. Furthermore, this direct comparison does not consider the true advantages of using paper or other flexible membranes as the support material for SERS substrates. That is, the inherent capabilities of paper to acquire and process samples can provide performance advantages that enable it to exceed the capabilities of conventional rigid SERS substrates.

III. PAPER-FLUIDICS: INTEGRATING SAMPLE PROCESSING INTO SERS DEVICES

While rigid SERS devices are well established as detectors, they do not provide any capabilities for sample acquisition or processing, adding to the challenges of utilizing them for routine chemical and biological analysis in the field. Over the last decade, a number of microfluidic devices have been proposed for SERS, but these devices may be as expensive to fabricate as some current micro/nano-fabricated SERS substrates, and bulky pumps and actuators are typically required for sample processing. Given the current cost and

Table 1: Paper SERS substrates, fabrication methods and characteristics

Fabrication method Detection Limit Enhancement Factor

Variability Reference

Vacuum Deposition (Vo Dinh et al.)

36 pg p-aminobenzoic acid, 2 pg carbozole and 14 pg 1-aminopyrene

Not reported 15- 20% [27], [28]

In-situ Growth (Berthod et al.) 0.7 pmoles aminoacridine Not reported 15%

[29-31]

In-situ Growth (Cheng et al.) Not reported 107 <8%

[41]

Drop-wise Deposition (Feng et al.)

Not reported 105 Not reported

[32-34]

Soaking (Singamaneni et al.) 140 pg 1-4 benzendithiol (on surface) and 0.5nM trans-1,2-bis(4-pyridyl)ethene (BPE)

5 x 106 15% [35], [36]

Soaking (Garnier et al.) < 1nM 4-aminothiophenol (4-ATP) 108 Not reported

[38], [39]

Screen printing (Qu et al.) 160 fM R6G 4.4 x 106 5-10% [50]

Inkjet Printing (White et al.) 4 pg (20 fM) BPE and 5 pg (10 fM) R6G 2 x 105 5-15% [42], [47]

Fig. 2. SEM images of paper SERS devices, reprinted with permission from (A) Lee, et al., [36], copyright 2011, American Chemical Society, (B) Ngo, et al., [39], copyright 2013, Elsevier, (C) Yu and White [42], copyright 2010, American Chemical Society, and (D) Mehn, et al., [40], copyright 2013, Elsevier.

100 µm 1 µm

A B

C D

10 µm


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usability challenges associated with microfluidic devices, a new paradigm has emerged in which microfluidic elements are designed as an integral part of paper analytical devices [51], [52]. In these elements, the wicking properties of hydrophilic paper or membranes are leveraged for liquid sample acquisition and transport. A variety of transduction techniques have been reported, including colorimetric detection [53–57], chemiluminescent detection [58–61], and electrochemical detection [53], [62–66]. A few recent reports have even demonstrated assays with the potential for diagnosis, including protein detection [58], [67], [68] and DNA sequence detection [69], [70].

The inherent flexibility and fluidic transport capabilities of paper can also be leveraged to add functionality to the low-cost paper SERS devices. While conventional rigid SERS substrates require manual addition of samples in small liquid droplets, paper SERS devices can perform sample collection with unprecedented ease. Lee, et al., were the first to highlight this, as they reported the use of paper SERS devices as surface swabs to collect trace analytes from surfaces [35]. The authors demonstrated the detection of 140 pg of 1,4-benzenedithiol (1,4-BDT). Our group demonstrated the use of inkjet-printed paper SERS devices as practical swabs by detecting down to 10 ng of the widely used fungicide thiram on surfaces (Fig. 3) [47].

In addition to collecting residue from surfaces, the wicking capability of paper makes it much more efficient for the collection of liquid samples as compared to the rigid SERS devices. Whereas with conventional devices the sample is collected into a sample container and then applied with a controlled deposition technique (e.g., pipetting), a paper SERS device can be dipped directly into the liquid sample in the field, where it can collect the sample and immediately subject it to analysis. Using a paper SERS dipstick with nanoparticles printed onto the tip – which is dipped directly into the liquid sample – we were easily able to detect 1 ppb of the fungicide malachite green after a 1-second dip [47]. By extending the time of the dip, additional sample volume is wicked into the paper device, which, as expected, increases the signal magnitude for a given concentration. In our experiments, the magnitude of the Raman signal for malachite green increased by a factor of three by dipping the SERS substrate for 30

seconds instead of just one second [47]. While sample acquisition by dipping or swabbing provides

an unprecedented ease of use, paper SERS devices also provide sample processing capabilities that are not possible with conventional rigid SERS substrates. In particular, paper devices enable separation of analyte molecules from a complex background, and the wicking properties of paper enable lateral flow concentration of analyte molecules, which boosts performance. To illustrate the use of flexible membrane SERS devices for analyte separation and identification, we have demonstrated detection of melamine spiked into infant formula [71].

For this application, polyvinylidene fluoride (PVDF) was selected instead of paper because of its high protein binding capacity. Infant formula spiked with melamine was spotted onto the PVDF device, which had been functionalized with silver nanoparticles by inkjet printing. Even at a concentration of 100 ppm, the Raman band for melamine could not be observed because proteins and other interferents in the infant formula foul the surface of the nanoparticles, thus preventing the melamine from interacting with the plasmonic surface (Fig. 4). After performing the chromatography using a mobile phase of 0.1% acetic acid for 1 minute, melamine is separated in the chromatogram (region III); a clear SERS signature of melamine is apparent, and very little background can be seen. The melamine is located near the solvent front due to its solubility in the dilute acid, while the larger protein molecules are localized where the sample was applied. We achieved detection of 5 ppm melamine in infant formula using this technique.

Separation of complex samples on paper SERS devices has also been reported by Abbas, et al. [37]. The authors constructed a star-shaped paper device that was loaded with gold nanorods by soaking. The “fingers” of the stars were treated with various concentrations of two polyelectrolytes, one positively and one negatively charged, to create a charge gradient around the tips of the stars. The gradient enables separation of complex samples by charge. The separation and detection of R6G that had been mixed into spinach concentrate is reported.

In addition to sample collection and cleanup, the wicking-based fluidic transport of paper also enables lateral-flow concentration of analyte molecules. Collecting a sample through a dip or a swab leaves analyte molecules scattered throughout the entire paper area. However, by cutting the paper device to form a tip, and by adding a volatile solvent to the end of the paper opposite the tip (e.g., by dipping), the analyte molecules can be concentrated at the tip by the continual movement of the solvent towards the tip. As an example, Fig. 5 shows the Raman spectra of R6G measured on SERS-active paper after swabbing a surface and then performing lateral-flow concentration of the swab using methanol [72]. The lateral-flow concentration results in a 24X improvement in signal magnitude.

We have also demonstrated the use of lateral-flow concentration following chromatography-based sample cleanup [71]. In this case, heroin is combined with a highly

Fig. 3. SERS signal obtained by swabbing glass slides with various amounts of thiram deposited on the respective surfaces. Reprinted from Hoppmann, et al., [47], copyright 2013, Elsevier.


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fluorescent dye (IR785), which is used to mimic cutting agents used in street drugs. Raman and SERS both fail to identify the heroin in this mixture because of the high fluorescence of the cutting agent. However, after chromatography on a paper SERS device, the Raman signal of the heroin is visible (Fig. 6), though the chromatographic separation has left the heroin diluted across a ~1 cm length of the paper. By cutting away the region of the paper at which the heroin is located and performing lateral flow concentration, the heroin is concentrated at the tip and the signal is significantly improved. We were able to repeatedly detect 25 ng of heroin spiked with the cutting agent using this technique.

It is clear from these results and the other reports discussed above that paper SERS devices, plasmonically functionalized through low-cost techniques such as printing, soaking, and in-situ synthesis, not only offer a dramatically lower cost solution for chemical analytics than conventional SERS substrates, they also offer advanced functionalities, including sample acquisition, sample cleanup, and analyte concentration; these features are simply not available from conventional

nanofabricated and microfabricated SERS devices. Furthermore, these advanced functions are possible without the need for pumps, actuators, tubing, or any of the other physical accessories required with typical microfluidic analytical devices. We expect that the combination of the unprecedented low cost, the high performance, and the

Fig. 5. SERS signals from swabbing a glass slide containing 24 ng of R6G before and after performing the lateral flow concentration. The SERS signal strength is improved by 24X due to the lateral flow concentration. SERS spectra are shifted for clarity. Reprinted from Yu and White [72], copyright 2013, Royal Society of Chemistry.

Raman shift (cm-1)

Distan

ce (m

m)

Sig

nal i

nten

sity

(co

unts

/s)

Melamine signature peak

I

II III

1 m

m

Before separation After separation

Solvent front

III

1 m

m

Silver NP ink on PVDF Solvent

flow

I II

Melamine peak

A

B

Fig. 4. (A) Infant formula laced with melamine (100 ppm) on PVDF membrane before and after chromatographic separation. SERS of regions I, II and III shows the dramatic improvement of the 695 cm-1 peak of melamine after performing the separation. (B) SERS chromatogram showing the location of melamine on the PVDF membrane. NP = nanoparticle. Reprinted from Yu and White [71], copyright 2013, Royal Society of Chemistry.

Separated heroin IR 780

Heroin reference

Raman shift (cm-1)

Distance (mm)

Heroin peak

A After separation

CCD saturation

10 mm

B

Solvent flow

Sign

al in

tens

ity (c

ount

s/s)

After concentration

Before concentration

trial 1

trial 2

trial 3

trial 1 trial 2 trial 3

10 mm

Solv

ent f

low

Heroin C

Fig. 6. (A) After chromatographic separation, the Raman signal for heroin is no longer obscured by the high fluorescence background of the cutting agent. (B) SERS chromatograph, showing the locations of the separated heroin and fluorescent cutting agent. (C) Following separation, the paper SERS device is cut to form a lateral-flow concentration device to focus the heroin at the tip. Adapted from Yu and White [71], copyright 2013, Royal Society of Chemistry.


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advanced functions of paper SERS devices will finally drive SERS beyond the research laboratory and out into the field for routine on-site chemical and biological analytics.

IV. PAPER SERS FOR BIOLOGICAL ANALYSIS

In general, SERS is capable of performing label-free detection of small molecules, as the spectral bands can easily be identified and distinguished. For macromolecules, such as large protein and DNA molecules, it is more common to employ SERS in a labeled immunoassay [73–76] or hybridization format [2], [20], [75], [77–84]. The labels, which are often fluorophores or other strong Raman scatterers, are referred to as Raman reporter probes (RRPs). As opposed to fluorescence-based transduction, however, the RRPs each generate a unique Raman spectral fingerprint upon laser excitation, which enables much denser multiplexing than fluorescence while utilizing only a single laser and a single filter set [2], [3]. These conceptual advantages make SERS an intriguing potential choice for a number of translational applications in molecular detection. Here we report a new result in which paper SERS devices are combined with polymerase chain reaction (PCR) and a hydrolysis probe to detect the presence of a genetic sequence. The amplification of targeted DNA sequences can be visualized in PCR by using a hydrolysis probe that hybridizes within the amplified sequence. A traditional hydrolysis probe (i.e., a TaqMan® probe) relies on a 5` fluorophore and a 3` quencher; when the probe is hydrolyzed (indicating the presence of the target sequence) the fluorophore is released from the probe and ceases to be quenched. Thus, detection is provided via the emergence of the fluorescence. However due to the broadband nature of fluorescence excitation and emission, these assays are typically limited to a few targets per reaction. SERS, which can improve the multiplexing density, has been previously demonstrated in combination with PCR [84–86], however these assays typically suffer from a combination of negative signal response, poor signal contrast, and complex probe design/processing steps.

In our PCR-based assay, a simple single-labeled DNA probe is utilized. This probe is designed with a 5` Raman reporter and binds to a complementary target DNA sequence which is bracketed by a specific set of primers. As the Taq DNA polymerase extends the primers, replicating the bracketed region, it will encounter the bound probe: the inherent 5` to 3` exonuclease activity of the Taq polymerase will then degrade the probe, releasing the Raman label from the DNA as shown in Fig. 7A. As described above, the paper SERS devices possess chromatography capabilities. Thus, PCR amplification of a targeted DNA sequence can be detected by leveraging the solubility differences between the released Raman reporter and the intact DNA probe. Following the PCR reaction period, the sample is deposited onto one end of a paper chromatograph, opposite to a tip onto which metal nanoparticles have been printed (Fig. 7B). After drying, the paper strip is dipped into a solution in which the reporter is soluble but the intact DNA probe is not (Fig. 7C). If PCR occurred (implying that the targeted sequence was present in

the sample), the highly soluble Raman reporter can be detected at the plasmonically functionalized tip using SERS (Fig. 7D). If PCR did not occur, no reporter can be identified at the SERS-active tip. Importantly, because of the narrow-bandwidth nature of SERS, multiple reporters can be detected simultaneously, offering a simple solution for multiplexed detection of genetic sequences.

A. Methods Custom DNA primers and probe (forward primer: GGA

TTA GCA GAG CGA GGT ATG TAG; reverse primer: GGT TTG TTT GCC GGA TCA AGA G; probe: TGG TAT CTG CGC TCT GCT GAA GCC AGT) were obtained from Integrated DNA Technologies (Coralville, IA). The probe is labeled with a 5` Carboxy Rhodamine 6G Raman label and a 3` phosphate group to prevent extension. Taq DNA polymerase with ThermoPol® buffer, pUC19, dNTPs and the restriction enzyme SspI were obtained from New England Biolabs (NEB, Ipswich, MA). The pUC19 was linearized with SspI according to the NEB protocol (with a 5 times excess of enzyme). Thermocycling was conducted using a 10 second melt step at 95° C, a 15 second annealing step at 68° C, and a 30 second extension step at 72° C. Cycling was repeated a total of 30 times and was followed by a final extension for 5 minutes at 72°C. Correct PCR product was verified by gel

Fig. 7. Depiction of the SERS-PCR assay for DNA detection using Raman probes. (A) PCR is performed using single-labeled probes. If the target is preset, the probe will be hydrolyzed during extension, releasing the Raman reporters. (B) The PCR reaction is applied to a dipstick with a printed SERS-active region at the top. (C) A separation is performed which allows the hydrolyzed probes (Raman reporters) to migrate to the top, while retaining the whole probes at the bottom. (D) A 532 nm laser and Raman spectrometer are used to read the SERS signal from the top of the dipstick.

A

B C

D

Hydrolysis PCR

·If target is present,

the primers and

probe will bind

·The PCR reaction

extends the primers

·Taq polymerase

hydrolyzes bound

probes, releasing

Raman reporters

Sample Application Separation

·Raman reporters

move to detection

region

·Whole probes,

being insoluble,

remain at bottom

SERS Detection

·532nm laser and

spectrometer used

to read signal of

Raman reporters

P

P

Printed Detection Region

Polymerase: Primer:

P

Probe:

Plain Paper


7

electrophoresis. For fluorescence optimization and validation of this assay, a

second probe (same sequence) was obtained with a 5` TEX™ 615 label. Fluorescence data was collected using an Olympus IX51 microscope, and the data was averaged across the width of the chromatography strips to produce plots of the fluorescence intensity as a function of distance traveled up the strips using Image-J (National Institutes of Health).

The silver colloid was synthesized using a simple reduction reaction. Silver nitrate, sodium citrate and dextran (average MW 150,000) were obtained from Sigma-Aldrich (St. Louis, MO). Briefly, 72 mg of silver nitrate was added to 400 mL of DI water (18.2MΩ) and brought to boil in a 500 mL Erlenmeyer flask. While stirring rapidly (such that the vortex just reaches the bottom of the flask), 80 mg of sodium citrate was added. The color slowly changed to a green-brown color. After 10 minutes the solution was removed and allowed to cool to room temperature.

The silver ink was formed by first centrifuging the silver colloid at 3,000g to concentrate the nanoparticles. After removing the supernatant, the pellet of nanoparticles was suspended in water to achieve a final concentration factor of 100X. Finally, the ink was created by adding dextran in water to achieve a final concentration factor of 50X with 2.5 mg of dextran per mL of ink.

For printing, the ink was injected into refillable ink cartridges. SERS-active regions were defined in the open source vector graphics editor Inkscape. Printing onto untreated chromatography paper (Whatman Grade 2) was conducted using an inexpensive Epson Workforce 30 piezo-based inkjet printer, as previously described [42].

SERS measurements were performed using a 532 nm laser (0.6 mW) for excitation and a Horiba Jobin Yvon LabRam ARAMIS Raman microscope for detection. The laser spot size was experimentally measured to be 12 µm. Measurements were rastered over a 200 x 200 µm region by the system (two passes per integration). An integration time of one second with 5 averages was used for all measurements. Immediately before measuring, 2 µL of 2.5% HCl was applied by pipette to the center of the printed region to protonate the Raman label and promote interaction with the nanoparticles. Using an

automated stage, measurements were taken at 6 200 x 200 µm locations per dipstick (equally spaced throughout the printed SERS-active region). Averaging the spectra from 6 points per dipstick helps to reduce the variability due to the uneven nature of chromatographic separation: for the 5 positive dipsticks shown here, the relative standard deviation, |σ/µ|, of the 1510 cm-1 peak height ranged from 15 to 29%.

For display, each dipstick’s 6 data sets were averaged together. A linear fit was subtracted to remove the contribution of florescence. A 5 point FFT smoothing filter was applied to the data before plotting.

B. Separation of Whole and Hydrolyzed Probe To demonstrate that a chromatographic separation can

discriminate between whole and hydrolyzed DNA probes, 20 µL PCR reactions using the TEX™ 615 label were pipetted onto the bottom of 8 x 58 mm strips of Whatman Grade 2 chromatography paper and allowed to dry. This paper was chosen as it has sufficiently small pores to promote substantial interaction with the whole probe while having sufficiently large pores to allow for good dispersion of the silver nanoparticles into the paper (resulting in high SERS enhancement). These strips of paper were then dipped into a solvent in a closed chamber and allowed to run for 20 minutes.

In Fig. 8, chromatographs are presented in which 60% ethanol is used as the solvent. Beyond the region of sample deposition, an increased fluorescence is seen reaching approximately 45 mm for the positive PCR sample (upper red line), indicating the movement of the released Raman reporter, while there is essentially no movement of the probe in the negative PCR sample (lower black line – intensity due to the inherent fluorescence of cellulose). The essentially on/off behavior exhibited by the positive/negative probes in the PCR reaction allows for clear discrimination.

C. SERS Detection of PCR product For SERS detection of genetic targets, 8 x 27 mm dipsticks

with 3 mm SERS-active regions printed at the top are used (Fig. 9). Similar to the chromatography experiments, the full 20 µL post-PCR sample is applied to the bottom of the dipstick with the printed end of the dipstick elevated by 4 mm to prevent unwanted wicking due to capillary action. After drying, the bottom 3 mm of the dipstick is dipped into a 60% ethanol solution in a sample vial for 20 minutes, with the top 3 mm of the dipstick emerging from the lid of the sample container. Allowing the SERS-active region to be exposed to air promotes an evaporation-driven concentration of Raman reporters into the detection region.

Representative SERS traces are shown in Fig. 9 for 5 positive (i.e., target included in reaction) and 5 negative (i.e., target not included in reaction) PCR runs. The clear on/off behavior observed in the fluorescence chromatography experiments translates to these SERS measurements, providing easily distinguishable SERS spectra for the positive and negative PCR dipsticks. The 3 characteristic peaks of R6G that are commonly used for identification and quantification are marked with asterisks (1310, 1360 and 1510 cm-1).

We have demonstrated the ability of this simple paper-based assay to clearly and consistently differentiate between positive

Fig. 8. Fluorescence intensity (averaged across the width of the strips) demonstrating the chromatographic differentiation between a positive PCR sample (top, red) and negative PCR sample (bottom, black). Inset shows the full range of intensity vs. distance data. The intensity of the right portion of the bottom trace is due to the inherent fluorescence of the paper.

Sample

applied

Solvent

flow

58mm

8mm


8

and negative PCR samples. By taking advantage of the narrow-band spectroscopic fingerprint provided by Raman labels, multiplexing well beyond that which is achievable using fluorescence becomes possible. We envision that, in combination with a low cost thermocycler and a commercially available handheld spectrometer, this approach could contribute to the development of DNA diagnostic tests enabling screening for a large number of targets within a single reaction.

V. CONCLUSION While numerous research reports have demonstrated the

power of SERS for chemical and biological analytics, a need persists for devices that are dramatically lower in cost with increased functionality. Recently, plasmonic paper has attracted attention as a low-cost method to perform SERS. Devices are fabricated through soaking, in-situ synthesis, or printing, making them far less expensive to produce than conventional nanofabricated devices. In addition, the flexibility and fluidic properties of paper add functionality to the SERS devices, including sample collection, molecular separation, and analyte concentration. Multiple reports were reviewed here in which paper SERS devices were used as sophisticated and highly sensitive chemical analysis tools. Furthermore, we reported here for the first time the use of paper SERS devices for the detection of PCR amplification. The chromatographic and lateral-flow concentration capabilities of paper are utilized to discriminate samples for detection of PCR amplification. This system will lead to simple methods to perform multiplexed target DNA sequence detection without requiring sophisticated laboratory facilities.

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Fig. 9. A simultaneous separation and concentration is performed with a post-PCR sample. Representative SERS spectra from 5 positive and 5 negative dipsticks are shown (spectra for the positive PCR samples are shifted vertically for clarity).

Solventheight

Lid

Sample

Printed NPsPositive PCR

Negative PCR

*

**

27mm

8mm

Raman Shift (cm-1)


9

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Eric P. Hoppmann received a B.S. in Physics from James Madison University in 2008. He then joined the Ph.D. program at the University of Maryland’s Fischell Department of Bioengineering. Mr. Hoppmann advanced to candidacy for his Ph.D. in 2011. His research interests include nucleic acids analyses, paper-based analytics, and surface enhanced Raman scattering. In 2012, Mr. Hoppmann co-founded the company Diagnostic anSERS and is currently serving as President of the company. Wei W. Yu received a B.S. in Physics and Math from the University of the South Pacific in Fiji in 2006. He then received an M.S. in Applied Physics from Southern Mississippi University in 2008. He then joined the Ph.D. program at the University of Maryland’s Fischell Department of Bioengineering. He completed his Ph.D. in Bioengineering in 2013 and is currently serving as a Postdoctoral Fellow at the Univesrity of Maryland. His research interests include paper-based diagnostics, metal nanoparticle chemistry, and surface enhanced Raman spectroscopy. Ian M. White received a B.S. in Electrical Engineering from the University of Missouri in 1997, and a Ph.D. in Electrical Engineering from Stanford University in 2002. He then joined Sprint’s Advanced Technology Laboratories, where he worked until 2005. At that time, Dr. White transitioned into the field of optical biosensors as a Postdoctoral Fellow in the University of Missouri Life Sciences Center. In 2008, Dr. White joined the faculty in the Fischell Department of Bioengineering at the University of Maryland.

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