Analytical Spectroscopy

Minute Paper #6 (10/25/2009) – Zhenpeng Qin
Title: Dynamic Nanoinscribing for Continuous and Seamless Metal and Polymer Nanogratings
http://pubs.acs.org/doi/abs/10.1021/nl902682d
By: Ahn et al.
Journal: Nano Letters

Fabricating nanoscale structures has attracted lots of research efforts pushed by the broad applications in optics, lab-on-chip, and bioindustries. The authors introduced a technique, namely dynamic nano-inscribing (DNI), to fabricate nano-grating patterns on a large area in a continuous and fast manner. To prove this idea, nano-gratings were fabricated with a variety of metal and polymer materials with room temperature and localized heating.

The authors illustrated the four steps of DNI process to distinguish DNI from nano-imprint lithography (NIL). The key difference is the gradually increased pressure over a very small contacting region in DNI, which enables DNI to fabricate continuous linear patterns over a long distance and work with hard materials. This was demonstrated by inscribing conductive ITO (Fig 2c), which is very difficult by traditional techniques. The authors shoed the viability and advantages of DNI by fabricating angled but continuous 200nm period gratings (Fig 2a) that was not possible by NIL, and square-shaped gold nanopatterns (Fig 2b) by two consecutive DNIs in orthogonal directions. If one looks closely to the square-shaped gold nano-patterns, there is some not well defined structure at the crossings of orthogonal nano-gratings. This comes from the mechanism of this technique, i.e. by deforming the material while not removing it.

Furthermore, the authors demonstrated the flexibility and advantages of DNI, including creating long and seamless grating patterns compared to roll-to-roll process, the integration of DNI with roll-to-roll process (roll-to-roll DNI, Fig 3a), and creating continuous and curved nano-grating patterns (Fig 3b) and on curved surfaces (Fig 3c). The speed of grating patterning by DNI relies on the plastic deformation, which is much faster than NIL relying on filling polymers into cavities on the mold. The authors showed that by locally heating the polymer substrate by Si mold conductive heating, deeper and sharper patterns can be created under the same conditions including the same applied force and web speed.

As a proof of concept, the authors created nanogratings on a variety of metals and polymer at room and locally elevated temperatures, including P3HT and PEDOT which have broad application in organic photovoltaics, organic thin-film transistors and LEDs, Nafion film (DuPont) which is an important material for fuel cell applications, and negative refractive index metamaterials (20nm thick silver on polycarbonate file, 200nm period grating with 30nm trench).

While this technique seems promising and could be used in a variety of different application, there are many unanswered questions and limitations. One of them is the ability to create complex micro- or nano-structures. This technique works well with the simple nanostructures, including straight and angled long gratings. But when it comes to squares, which is a little more complex, the structure is not well defined, characterized by the scratches in the nano-grating crossings (Fig 2b). One can imagine that if the structure becomes more complex, the application of this technique is limited. Another limitation is that the gratings always have a dome-shape. Is there any way to further improve this, for example by varying the shape of the mold?

Title: Inverse-FluorescenceCorrelation Spectroscopy

Journal: Analytical Chemistry

Author: Stefan Wennmalm et al

Link: http://pubs.acs.org/doi/full/10.1021/ac9010205

In this paper, the authors attempt to observe the fluorescent signal from the medium surrounding the analyzed particles instead of observing the signal from the particles themselves. It’s quite a novel idea to analyze the solute in the solution and avoid labeling the solute of interest. Techniques involved in this paper are fluorescence correlation spectroscopy (FCS), argon ion laser, avalanche photo diodes, correlator board and CONTIN algorithm.

Signal reduction is observed from Figure 2 and the linear relationship between amplitude and number of particles is obtained which shows that this method is applicable for the detection. Size dependent detection is also investigated in which particles of 4 different sizes are compared. Their solutions show different fluorescent intensities which renders this method a good way for size analysis. In addition, an intensity function of particle number, particle size and detection volume is developed to demonstrate the relationship between intensity and properties of solution.

However, several problems still need to be addressed in this paper. First, the fluorescent signal changes in figure 2 are not very convincing especially for A and B. It can be doubted that this method can not be applicable to the particle of size less than 200nm; Second, the particle size distribution should be given because the size of the synthesized particles cannot be so accurate and should fall within a range; Third, beads are different from molecules. Beads are sphere but biomolecules may have a variety of shapes and different density or electron distributions which may have some effects on the signals. So shape experiment can be conducted and in addition, shape parameters can be added into the equation 3 to determine the relationship more accurately. For preliminary experiment, simple shapes like rod, disk and sphere can be included. Forth, different solvents may be used to find the best one. The signal is based on the change of the environment so properties of the solvent may influence the signal greatly. Solvent with high fluorescent signal can be suggested to increase the signal to ratio.

I think one limitation for this method is that there are so many factors to influence the medium surrounding molecules so it’s really hard to get the most accurate information we need. On the contrary, direct detection from the molecules is easier to get the information because variant factors here are much less.

Minute Paper #7 – 10/30/09 – Michelle Henderson

Title: A Straightforward Method for the Colorimetric Detection of Endogenous Biological Cyanide

Authors: Christine Mannel-Croise et. al.

Journal: Analytical Chemistry

A colorimetric detection to be used outside of the laboratory was developed to detect biological cyanide in cassava root, a major carbohydrate source in South America. The release of cyanide from cyanogenic glycosides in plants by enzymes during digestion (in animals) is a natural defense mechanism of plants. First, the authors studied the kinetics of the substitution of Co(III)-bound chemosensor with cyanide to determine the rate of the reaction (about 3 seconds) as well as a provide a clue as to the mechanism (most likely associative). Then the release of biological cyanide from the cassava root was studied via enzymatic and acid hydrolysis liberation in order to validate their method against the biological mechanism. And last, the method was tested for application in food processing. Simple UV-vis spectra were obtained using a spectrometer and quartz cell for the kinetics experiments, and diffuse reflectance UV-vis (DRUV-vis) spectroscopy was used to examine the substitution of the chemosensor with cyanide in the cassava root.

The most obvious advantage of this technique is that it can be used outside of the laboratory in the absence of spectroscopic instrumentation in order to obtain a simple positive/negative result. The substitution of the chemosensor with cyanide can be seen quickly (about 3 seconds) and with the “naked eye” as the color of the solution changes from orange to violet. This was applied to food processing where the authors determined if boiling or washing of the cassava root were acceptable methods in order to rid the food of cyanide. It was illustrated that no cyanide could be detected after 4 washings, but still 65% of the cyanide remained after boiling the root for 1 hour. Also, the only sample preparation of the root included homogenizing and then subsequent acid hydrolysis.

The authors admit that this is not a very quantitative method. They were able to estimate the amount of cyanide found in cassava root samples (122-265 mg/kg), but did not report an LOD. This large range for the amount of cyanide detected in the root samples was reported, but do not address as to why it is so large. It appears as though some measurements are only duplicates, so more measurements need to be taken to strengthen their conclusions. The authors would also need to specify if these samples were from different cassava plants, if they were taken from different parts of the plants, and if different samples were purchased at different times from different vendors (different sources). This may account for some of the variation and give insight as to where and why cyanide concentrations are higher in some specimens rather than others. It would also be helpful if the authors had tested several different food samples and reported these amounts with their respective LODs. They may or may not have found that different sources have different interferences, pigmentation or other chemicals, that may interfere with their method causing it to be not as ready for the widespread application that they claim.

In addition, the biological samples must be colorless so that they do not interfere with the calorimetric detection, which severely limits what can be tested. In order for widespread application of this technique, some sort of washing or extraction method must be developed to rid the samples of any pigment. This would come at a great disadvantage because cyanide will be washed away with any general washing, and this will add to the sample preparation process, which was a great advantage of this technique. Also, if this method can be applied to crude plant samples, why could it not be applied to animal tissue samples as well?

Minute Paper- 2009.10.30 - Antonio Campos
Title: Thin-Layer Chromatography/Laser-Induced Acoustic Desorption/Electrospray Ionization Mass Spectrometry
Authors: Sy-Chyi Chemg, Min-Zong Huang, and Jentaie Shiea
Journal: Analytical Chemistry
Link: http://pubs.acs.org.floyd.lib.umn.edu/doi/abs/10.1021/ac901514c

In this paper, the authors investigate the use of thin-layer chromatography (TLC) plates for laser-induced acoustic desorption electrospray ionization mass spectrometry (LIAD/ESI/MS). The benefits of using this technique (shortened to TLC/LIAD/ESI/MS) are the separation of inorganic and organic compounds can be achieved, both volatile and semi-volatile compounds can be analyzed, and sample switching is rapid. Analytes of interest in this paper are dye standards, drug standards, and rosemary essential oil. Techniques used in this paper are obvious from the above abbreviation that is thin layer chromatography, laser-induced acoustic desorption, electrospray ionization, and mass spectrometry.

The authors show that using a reversed phase (C18 gel) TLC plate or a normal phase (silica gel) TLC plate detection of the analytes can be achieved. However, simply using an aluminum backed TLC plate for LIAD proved to be difficult due to the fact that the Nd:YAG laser used to ablate the surface of the TLC plate could not be achieved with laser light hitting the aluminum side of the plate (i.e. the back of the TLC plate). However, when the authors directed light to the surface of the TLC plate the silica gel/C18 gel beds did ablate. To circumvent this limitation, the authors taped a glycerol coated glass microscope slide to the back of the TLC plate and laser light was directed to the back of the plate. Through the acoustic wave generated by the impinging light, the top of the TLC plate was ablated, with a laser power density greater than 2.1 x 109 W/cm2. Laser power density (PD) is defined as laser energy divided by pulse duration times the spot area (PD = laser energy/ (pulse duration x spot area).

One obvious advantage of this technique is that shining light on the back of a TLC plate to introduce the sample into the ESI plume does not affect photosensitive analytes. The analytes of interest include: FD&C Red no. 3, LSD (lysergic acid), and rosemary essential oil. The first two are part of a standard dye and drug group, while the third is a proof of concept for this technique that represents the complex mixtures that can be analyzed. The results for the dye and drug standards yield expected m/z ratios, but the rosemary essential oil yielded complex results due to the large amount of analytes present in the unprocessed sample (up to 20 spots on the TLC plate). Due to limited databank entries for rosemary essential oil, the authors can only tentatively assign three m/z peaks to α-pinene, thymol, and camphor.

The proof of concept samples chosen for this paper, are obvious signs of where the authors intend to direct their research. One can envision, using a TLC plate to separate an unknown sample found by forensic scientists in order to analyze the sample via mass spectrometry. The authors repeatedly express that the sample preparation is under ambient conditions, and damage to photosensitive samples will not be occur. Perhaps, this can be used to introduce samples into a lab on a chip set up for field measurements by scientists. One area that the authors do not discuss is they do not express the efficiency of the LIAD process for introducing analytes into the ESI plume, and subsequently into the ion trap MS. The mechanism for the introduction into the ion trap is based on absorption of the analyte by the droplets from the ESI capillary, but the authors do not express how efficient this process is. Calculating the efficiency of this process is complicated, however the ability to do so would offer a technique that could offer quantitative and qualitative data.

Minute Paper #5 (10/30/2009) – Hyungsoon Im

Title: Large single-molecule fluorescence enhancements produced by a bowtie nanoantenna
By: A. Kinkhabwala et al., from University of California at Berkeley (W. E. Moerner’s group)
Journal: Nature Photonics, published 18 Oct 2009

The authors made a bowtie shaped nanoantenna which creats highly enhanced local fields for single fluorescent molecule detection. They measured the fluorescence brightness enhancements for single fluorescent molecules as a function of bowtie gap size. They claimed they could get a high enhancement factor (EF) of 1,340, which is ten times higher than reported previously. The single molecule detection is confirmed by discrete blinking and eventual photobleaching of 50% of the total signal that can be attributed to a single molecule’s dynamics. In their finite-difference time-domain (FDTD) calculations, the total fluorescence enhancement ratio is 1,690 whose value is in good agreement with the maximum experimentally measured enhancement factor of 1,340. Also the simulation shows that the enhancement is high in the nanogap region, but quickly decreases above the metal surface. It is interesting that the maximum fluorescence enhancement occurs at the centre and decreases closer to the metal surface. It is because of lower quantum efficiencies arising from increased ohmic losses at the surface.
The results shown in the paper are interesting and their analysis is logical, but there are a few more things that would be better to be studied more in detail. They present data with error bars averaged by 5 highest signal spots and the EFs are in a wide range from a few tens to above a thousand. It is because molecules are randomly distributed in a solution and a few selected molecules placed near or in the nanogap would give such a high EF while many molecules placed elsewhere gives different values of EFs. They may not use self-assembled monolayer because they can be quickly photobleached near the metal surface. In another way, however, if they coat the surface with a thin layer of polymer linker or dielectric layer that increase the spacing between metal surface and fluorescent molecule. Another thing they may need to explain is that they claimed the biggest EF (~1,340) they have is ten times bigger than previously reported values (would be around 100~200). But the EFs they measured from single triangles range up to almost 500 in figure 2. So without bowtie nanogap, they already have 2~3 times bigger enhancement than others, and the nanogap only gives 3 time further enhancement compared to the single triangles. I guess they have some assumptions not mentioned in the text well, but it would be better if they can discuss it in detail.

Minute Paper #7 – (10/30/2009) – Si Hoon(Sean) Lee
Title: High Throughput single molecule detection for monitoring biochemical reactions
By: Paul I. Okagbare et. al.
Journal: Analyst Supporting Material:
In this paper, Okagbare et. al. develop the high throughput single molecule (ss-DNA with an intercalating dye) detection system with an electron multiplying charge coupled device (EMCCD) in a frame transfer mode (FTM). PMMA microfluidic device is used to accommodate multiple channels for a fluorescent labelled ss-DNA flow. Laser Diode is used as a light source and filtered using a laser line filter. The multiple images obtained from the high-spped FTM-EMCCD make it possible to detect the single molecule level concentration. The authors incubate the ss-DNA with the 10pM diluted intercalating dye (Syto-63) and claim the probability of single molecular occupancy is 0.01 and that of double occupancy is 0.0001. During the experiment they injected the diluted ss-DNA with electrokinetic microfluidic sytem.
In this paper, the authors claim that they realized the highly sensitive imaging system (100pM) with the high-throughput (4.02x10^5 molecules/sec) manner under their assumption-very low probability of double occupancy. For the high-throughput, they claim 5x objective is better to maximize the throughput due to the large field of view (FoV) but admit this prevent the high resolution image. Although, they claimed the high-throughput can go up to 1.78x10^6 theoretically when they use 1um channel width and spacing, it looks very challenging because the resolution of this fluorescent image is quite low (30um level) with the 5x objective. It seems like they are struggling to solve two paradox situations- high-throughput and low resolution, high-resolution-low throughput. For single molecule detection, it would be better not just to image the low concentration molecule but to show that they can isolate or manipulate the molecule. Highly parrallel microarray system may solve this high-throughput paradox problem instead of applying highly serial microfluidic system.

Link for the minite paper 07

High Throughput single molecule detection for monitoring biochemical reactions