Monday, 7 December 2015

Nanotechnology Made Easy and Cheap! Hydrophobic Carbon Nanoparticle film from Candle Soot


Around this time of year, with the holidays of Christmas, Hanukkah, Yuletide and end of the year celebrations in general there just seems to be tons of candles everywhere! Wax candles are just cylinders made up of hydrocarbons, which is the fuel that makes them burn and they can burn for a surprisingly long amount of time! When it comes to storing chemical energy you can't do much better than hydrocarbons. Hydrocarbons, in case you don't know, consist of molecules made up of carbon and hydrogen (The name is a dead giveaway!). When a candle burns, oxygen in the air reacts with the hydrogen to form water and with the carbon to form carbon-dioxide. This chemical reaction breaks the chemical bonds hydrogen has with the carbon and releases energy which in turn powers a complex plasma that we know as a flame.


A candle burns hydrocarbons made up of hydrogen and carbon with oxygen to form water and carbon-dioxide


The hydrogen of the hydrocarbon molecule tends to react slightly quicker so there is a region of the flame with small particles of pure carbon. So when you put something cold in this region these will condense on the surface forming black soot. (Moreover, putting something in the the way of the flame this will "starve" the flame of oxygen and unbalance the reaction towards producing more soot by La Chatelier's Principle - see further in the article)


The hydrogen buns more quickly than the carbon, so there is an area of the candle which is carbon-rich. So if you put something cold in the way, that also "starves" the flame of Oxygen, the carbon will condense as soot.


If you look at this soot on a microscopic scale it is extremely rough. This is because the soot is made up of lots of different sizes of carbon nanoparticles.The carbon particles themselves are quite dark already but the roughness makes it even darker as any light that reflects from one particle will often hit another and get absorbed.

Soot is dark not only because the nanoparticles are dark, but also because the soot is made up of lots of different sizes of carbon particles. This makes the surface rough so that most of the light that is reflected will hit another particle and be absorbed.

The carbon nanoparticles by themselves only repel water slightly. However, the different kinds of soot nanoparticles close together make the layer quite hydrophobic, so the water will only barely touch the very highest bits of the soot. The surface tension of the water can then support the water in between these peaks and you get a layer of air trapped between the water and the soot.


The soot particles repel water slightly and because it is so rough, surface tension will hold the water away from the surface. This traps a layer of air between the soot and the water.


Now we can test for the overall hydrophobicity. How? Well, the surface of the water will now reflect light very well due to an effect called total internal reflection. This happens because light travels more slowly in water than in air and whenever light changes it's speed it will be refracted due to conservation of momentum. When it moves from a travelling in a slow material (like water) to a faster one (like air) it is bent towards the surface. This is positive refraction. So, in effect, the black hydrophobic soot will become a silvery mirror in the water! 



The light is totally internally reflected at small angles, so the surface looks silvery and behaves like a mirror.

Here is a video I produced demonstrating all of this, where I grow a nanoparticulate carbon soot film on a coin from my wallet! -



So we have tested our candle soot layer is hydrophobic!

There are ways to improve the efficiency of this simple technique. For example, by "starving" the fire of oxygen, i.e. by having the candle within a tall candle holder, we can produce more soot than if it was out in the open. This happens because of a thing  in chemistry called "Le Chatelier's Principle"


So if we want more soot, C on the balance, we have to increase the amount of hydrocarbon fuel we have to burn relative to the amount of oxygen we have to consume. The key point here is "hydrocarbon fuel relative to oxygen" - we could always have a bigger candle of course but if we use the same candle and instead decrease the amount of oxygen, the balance will tilt down on the side of the reactants (think less O2, less relative weight on the balance) and this will tip the products side upwards. If we used half the oxygen as in the balanced equation above, what would happen on the reactants side? Well, we have half the amount of water produced but the carbon dioxide would remain the same. What would have to increase then to restore the relative weights is the carbon, C. In other words the hydrogen, which is very reactive remember, would more readily than before combine with the deprived oxygen and leave the carbon atoms behind- hence we get more soot!

Candle soot can then be utilised in a budget approach to self-cleaning surfaces that are both water and oil repellent.(a property collectively known as omniphobicity). There are many areas where such self-cleaning surfaces are needed. Examples include everything from windows, screens, optical devices such as fiber-optics and the glasses people wear. 

Soot is not very stable however, so if we were to grow the carbon soot on glass for example it would need to be coated with a silica shell. This could also be done with SiO2 nanoparticles of course which are commercially available. Glass can also be calcined, turning the sooty black coating into a transparent film on the surface which is more stable still. 

The simplicity of the approach is what particularly appeals to scientists - as it lies in the true vein of what it is all about; looking at something anew that was right under your nose all along. 

The positive fact of this is that non-specialists can make materials of this type and then investigate their properties is clearly something that's going to further advance this interesting area of research into new frontiers.




More Info for the Biotechnology Inclined...


Carbon nanoparticles (CNPs) have have great potential in health and environmental applications. They also could form the building blocks for future biomedical nanodevices because of their fascinating photoluminescent properties, not to mention having the potential to serve as nontoxic replacements for traditional heavy-metal-based quantum dots. (Ref1)

Flame and fluorescence: Water-soluble, multicolor fluorescent carbon nanoparticles can be prepared by refluxing candle soot with nitric acid (picture credit Wiley Online Library).

CNPs are nano-crystalline with predominantly graphitic structure and shows green, yellow, and red fluorescence under UV exposure. CNP-based fluorescence bioimaging probes are of key interest in cell imaging applications. CNPs can enter into a cell with no further functionalization and the fluorescence property of these particles is simple to study for calibration.

CNPs, prepared from candle soot, can then be further treated by a simple reflux rection with a strong oxidizing agent, such as HNO3 (Nitric Acid) or H2SO4 (Sulfuric Acid). (Ref2) As far as one can tell from the literature online, CNPs They can also be treated in the same way as Commercial Multi-Walled Carbon Nanotubes, MWCNTs, namely with either concentrated sulfuric acid and nitric acid mixture with ratio 3:1 or with a 0.1 M KMnO4 (potassium permanganate) via reflux, stirring and ultrasonication and subsequently separated by a single centrifugation. Apparently treatment in a acidic KMnO4 solution in reflux is the best for the reflux reaction kinds of treatment, and is the most attractive and safe method overall. However, the most precise treatments have in fact shown to be with superacids, such as HF/BF3, however the danger involved in using such solutions is what makes it less attractive. (Ref3)

The CNPs produced from candle soot typically have a size of 20–100 nm, height of 3.0 nm, a flourescence lifetime of 7.31 ns ± 0.06 ns and quantum yield of ∼1.7%. 

Further studies demonstrate that: 

(1) CNPs exhibit excellent stability in biological media and their luminescence intensity does not change with ionic strength or pH in the physiological and pathological range of pH 4.5–8.8.

(2) CNPs can act as electron donors and transporters and porphyrin can assemble onto the surfaces of CNPs through electrostatic and π-stacking interactions to form porphyrin-CNPs supramolecular composites. One of the most important porphyrins is heme, the pigment in red blood cells, a cofactor of the protein hemoglobin.

(3) CNPs have strong intrinsic peroxidase enzyme-like activity, which is very important. Peroxidase is an organic enzyme that transfers oxygen from hydrogen peroxide (H2O2) to other readily oxidisable substances.This gives it an amazing set of applications.



It is important,for example, for producing instant color-based glucose tests. Standard glucose color tests that use peroxidase working in sequence with Glucose Oxidase enzyme, GOx. GOx converts Glucose into H2O2 and Gluconic Acid. Then, the H2O2 product is reduced in catalysis by horseradish peroxidase, HRP, in conjunction with an ABTS colorimetric assay which, when it recieves a donor electron from the H2O2 reduction (i.e. becomes oxidised), changes color. There are already ways to assist this reaction, for example gold nanoparticles, AuNPs,  have been used to carry the HRP enzyme in solution to assist the catalysis of H2O2, in the vein that nanoparticles can help assist the spatial coupling between enzymes and thus increase the speed of catalysis. 



However, if we imagine that the nanoparticles used are in fact the catalyst itself, in effect using our CNPs to replace the HRP-AuNPs, not only is the spatial coupling increased but the surface area of the active sites is also increased, unlike in standard nanoparticle-enzyme bonding where the surface area of the active sites is in fact decreased due to the enzyme having to be bonded to the nanoparticle surface. It also saves the step of having to bind the nanoparticle and enzymes together in the first place, not to mention saving the expense of creating AuNPs and HRPs separately.

In any case, the colormetric tests will work more or less the same, simply needed different calibrations as the concentration of the glucose can be related to the intensity of color change of the ABTS. The more the intensity of the change, the higher the concentration of glucose. A simple color chart can be used to "read" the concentration of the glucose.

Based on the intrinsic peroxidase activity of CNPs, simple, cheap, and highly selective and sensitive colorimetric and quantitative assays can be developed for the detection of glucose levels for biosensing applications, in blood or food for example.

Standard peroxidase enzyme is also used to catalyse the oxidation of luminol to 3-aminophthalate via several intermediates. The reaction is accompanied by emission of low-intensity light at 428 nm. However, in the presence of certain chemicals, the light emitted is enhanced up to 1000-fold, making the light easier to detect and increasing the sensitivity of the reaction. The enhancement of light emission is called enhanced chemiluminescence (ECL).

For example, horseradish peroxidase enzyme (HRP) can be tethered to an antibody that specifically recognizes the molecule of interest. This enzyme complex then catalyzes the conversion of the enhanced chemiluminescent substrate into a sensitized reagent in the vicinity of the molecule of interest, which on further oxidation by hydrogen peroxide, produces a triplet (excited) carbonyl, which emits light when it decays to the singlet carbonyl. Enhanced chemiluminescence of this kind allows detection of minute quantities of a biomolecule. Proteins can be detected down to femtomole quantities, well below the detection limit for most assay systems.



In recent years the technique of marking neurons with the enzyme horseradish peroxidase has become a major tool in the detection of the smallest quantities of proteins - this is of particular importance in Alzheimer's research and Motor Neuron disease in which faster detection of even the smallest imbalances offer the greatest probability of successful treatments being developed and implemented and in the same vein of cancer research and treatment. 

Bioelectric cathodes can also be created based on co-immobilization of glucose oxidase enzyme (GOx) and horseradish peroxidase (HRP) onto a carbon nanotube modified electrode. In the presence of O2, GOx converts Glucose into H2O2 and Gluconic Acid. Then, during the reduction of H2O2 catalyzed by horseradish peroxidase, HRP, a direct electron transfer occurs between the carbon nanotube electrode and the peroxidase heme group. Hence it might be possible to fix carbon nanotubes onto carbon nanoparticles which, when "dipped with a GOx/HRP, will generate a voltage in the presence of glucose and oxygen. This could create nanoscale devices powered by glucose in the presence of oxygen wich would be very important in the field of biomedical implants. (Ref4)

In addition to biomedical applications, peroxidase is one of the enzymes with important environmental applications. This enzyme is suitable for the removal of hydroxylated aromatic compounds (HACs) that are considered to be primary pollutants in a wide variety of industrial wastewater



For example, phenols, which are important pollutants, can be removed by enzyme-catalyzed polymerization using horseradish peroxidase (HRP). Thus phenols are oxidized to phenoxy radicals, which participate in reactions where polymers and oligomers are produced that are less toxic than phenols. It also can be used to convert a variety of toxic materials into more harmless substances. 

There are also many investigations about the use of peroxidase in many manufacturing processes like adhesives, computer chips, car parts, and linings of drums and cans. Other studies have shown that peroxidases may be used successfully to polymerize anilines and phenols in organic solvent matrices.

All of this taken into account, discovering a way to cheaply produce nanoparticles and impliemnted them into controlled structures, such as in a "Lab-on-a-chip", to prevent any harmful externalities such as uncontrolled runoffs or contamination, we can make devices that perform similar functions to enzymes such as peroxidase, can radically enhance the development of solutions to many of the problems facing the world today which can be solved by combining the fields of biotech and nanotech.




References: 

(Ref1) Fluorescent Carbon Nanoparticle:
Synthesis, Characterization and Bio-imaging Application
S.C. Ray(a),*
, Arindam Saha, Nikhil R. Jana*
and Rupa Sarkar
Centre for Advanced Materials, Indian Association for the Cultivation of Science, Kolkata-700032 (India) 

(Ref2)- Bioengineering Applications of Carbon Nanostructures 

(Ref3)-Springer Handbook of Nanomaterials edited by Robert Vajtai

(Ref4) - Glucose Oxidase/Horseradish Peroxidase Co-immobilized at a CNT-Modified Graphite Electrode: Towards Potentially Implantable Biocathodes
Authors
Dr. Wenzhi Jia,
Dr. Chen Jin,
Dr. Wei Xia,
Prof. Dr. Martin Muhler,
Prof. Dr. Wolfgang Schuhmann,
Dr. Leonard Stoica
First published: 1 February 2012


Gold nanoparticles-based nanoconjugates for enhanced enzyme cascade and glucose sensing
Dongdong Zeng,ab   Weijie Luo,b   Jiang Li,*b   Huajie Liu,b   Hongwei Ma,a   Qing Huangb and    Chunhai Fan*b