The in- vention of STM and AFM opened a wide window to study the atomic-scale phenomena at the liquid-solid interface. Experiments showed that if the liquid is clean, atomic resolution can be readily achieved. Furthermore, the well-established methods of electrochemistry can be applied to change the surface and the adsorbed atoms and molecules. The pioneering works in this field [19, 20] showed a great potential of operating STM in liquids by using a two-electrode system: the substrate and the STM tip.
Later on, by combining STM with the standard methods of electrochemistry [21, 22], a four-electrode system was introduced [23, 24], which provides a powerful, general and convenient method to study the liquid-solid interface and a large number of electrochemical processes down to atomic level.
The stan- dard electrochemistry cell contains three electrodes [25]. The working elec- trode WE is the substrate under investigation. The four-electrode electrochemical cell with STM. The standard elec- trochemical cell has three electrodes: the working electrode WE , the reference electrode RE , and the counter electrode CE. By ramping the potential of the working electrode back and forth, a cyclic current-potential curve can be generated. The STM tip is the fourth electrode, to provide tunneling with the working electrode.
To prevent the harm- ful effect of oxygen, the entire cell is placed in argon atmosphere. Original figure by courtesy of J. See [26] for details. RE provides the reference potential, with insignificant electrical current flow. Typically, a saturated calomel electrode SCE is used as the ref- erence electrode [25]. The counter electrode CE supports the Faradaic current to or from the working electrode.
A routine experiment in electro- chemistry consists of a cycle to ramp the potential of the working electrode up and down with regard to the reference electrode. The Faradaic current is recorded as a function of time, thus also a function of potential. The cyclic current-potential curve, the so-called cyclic voltammogram, contains a rich body of information regarding the electrochemistry at the liquid-solid interface. The tip should be covered with an insulating film except on the very end of the tip.
The technique of making such a tip is described in Section Oxygen often reacts with the adsorbates of interest, especially biological molecules. To improve the cleanness of the liquid-solid interface, oxygen should be removed from the electrolyte. An effective method is to enclose the entire system in an inert-gas atmosphere, such as nitrogen or argon. To reduce the vibration caused by the flow of inert gas, a buffer bottle and two valves are installed to limit the flow rate, see Fig 1.
As in all STM-related experiments, an important issue is to start with an atomically flat, well-defined and well-understood substrate surface. The Au surface is an excellent substrate for the operation of STM in liquid. Small pieces of single crystal gold can be generated by flame annealing using a H2 flame.
The single crystal, typically a few mm in diameter, shows different crystallographic planes on its surface. After identified, a small piece of gold chip is cut off from the sphere then mounted on a sample holder. It provides a calibration of the x, y scales. Original images by courtesy of J. This provides a natural calibration for the x, y scales, and the identification of the crystallographic orientations of the gold surface with regard to the scan directions.
If the tip is in good condition, atomic resolution is achievable, see Fig. In the UHV environment, in order to change the surface reconstruc- tion, depositing or removing a layer of atoms or molecules at the surface, time-consuming and often irreversible annealing and vacuum evaporation processes are required. In an electrochemical cell, those processes could be accomplished within seconds by simply changing the potential, and are almost always reversible.
For example, the potential-induced reconstruc- tion has been observed on all three low-Miller-index gold surfaces. The second peak around 0. The very narrow peak means that the ordered adlayer is formed within a very small potential range. The upper half, recorded at a potential of 0. The lower half, recorded at a potential of 0. In order to observe large biomolecules such as nucleotides and proteins with STM or AFM in aqueous solutions, a key step is immobilization: to adsorb the molecule on a flat surface without changing its functions.
A voltammogram and an STM image of Au in 0. It starts at The very sharp peak at around 0. An order adlayer of sulfate is formed. Adapted with permission from Kolb [24]. The thiols, the or- ganic molecules with a thiol functional group -SH, can form a strong S-Au bond on gold surfaces about 1 eV , leading to self-assembled monolayers.
The other functional groups in that molecule, such as -OH, -CH3 , -CHO, could provide immobilizing anchors to the molecules of interest. Organic molecules containing carboxyl -COOH and amine -NH2 functional groups are important for immobilization of biological macromolecules.
Among the thiols, cysteine is of particular interest [27]. Cysteine is a particularly important building block in the formation of human hair, fin- gernails, skin, and wool, and a ligand in many metalloproteins.
Cysteine is one of the two amino acids which contain sulfur, and the only amino acid which contains a thiol group. A self-assembled monolayer of cysteine on gold surface provides both the carboxyl group and the amine group for the immobilization of biological macromolecules, especially proteins.
Self-assembled monolayers of cysteine are formed by exposing Au to a dilute solution of cysteine in 50 mM aqueous buffer electrolyte of am- monium acetate CH3 COONH4 [27]. The advantage of using ammonium acetate is that pH is around 4.
Self-assembled monolayer of cysteine on Au The sulfur atom is bonded to the Au surface at the hollow site. Reproduced with permission [27]. Copyright American Chemical Society.
A typical STM image of a highly ordered monolayer of cysteine thus obtained is shown in Fig. A structural model derived from the experiments is shown in Fig. A ball-and-stick model of cysteine is shown in Fig. As shown, the sulfur atom is bonded to a hollow site of the Au surface. The lateral interactions of cysteine finally generates an ordered pattern. In other words, each dot would contain in its area about 1, atoms.
At that time, such a dense writing was a fantasy. The invention of STM has completely changed the scenario. Using STM as a writing tool, features made of single atoms can be generated, far ex- ceeded the goal of Feynman. With such a density, the publications of the entire US Congress Library could be written on the head of a pin. The basic operation of atom manipulation is to use the STM tip to move an adatom from an initial position to a new position on a substrate, as shown in Fig.
At the beginning, the atom for the designed structure is deposited randomly on a substrate. Each atom is then an adatom on the substrate at a random position. In order to move an adatom to another stable location, an activation energy E must be applied to lift it across a ridge to reach another stable position.
There are three steps for a move, see Fig. As a result, the tip moves towards the adatom. A partial chemical bond is formed. When the chemical bond energy equals the barrier energy, the tip should be able to pull the adatom over the ridge. Step B is to move the tip sideways to pull the adatom to a desired location. Step C is to gradually decrease the set tunneling current. As a result, the tip moves away from the adatom and leaves it at the new position.
During the process of atom manipulation, neither the interaction energy between the tip and the adatom nor the distance between the apex atom of the tip and the adatom is known. The pioneers of atom manipulation found from experience that this can be controlled by tunneling conductance, or its inverse, the tunneling resistance. The threshold interaction energy to allow the tip to pull the adatom over a diffusion barrier corresponds to a threshold conductance, or equivalently, a threshold resistance [28, 29].
There is a general and fundamental relation between interaction energy and tunneling conductance, which will be discussed in Chapter 5. An example of the writing process is shown in Fig.
Those Chinese characters were written using Ag atoms on Ag surface using the above process. At each stage of the manipulation, an image could be taken to show the progress and to figure out the next steps to be executed. The size of each dot is less than 0. The basic steps of atom manipulation.
First, position the tip to above the adatom to be moved. Then, following the three steps: Step A, gradually increase the set tunneling current to move the tip towards the adatom, until the interaction energy between the tip and the adatom reaches the diffusion activation energy, the energy required to move the adatom across the ridge between two adjacent stable positions.
Step B, pull the adatom to a desired location. Step C, gradually decrease the set tunneling current to move the tip away from the adatom. Writing Chinese characters using STM. The lateral manipulation technique allows the exact placing of single atoms on desired atomic sites.
An assembly involves not only the movement of single atoms but requires also many repair and cleaning steps until the final structure is completed [1]. Courtesy of K. Braun and N. What are the most central and fundamental problems of biology today?
They are questions like: What is the sequence of bases in the DNA deoxyribonucleic acid? What happens when you have a mutation? How is the base order in the DNA connected to the order of amino acids in the protein? It is very easy to answer many of these fundamental biological questions; you just look at the thing! You will see the order of bases in the chain; you will see the structure of the microsome. Unfortunately, the present microscope sees at a scale which is just a bit too crude.
Make the microscope one hundred times more powerful, and many problems of biology would be made very much easier. The scanning electron microscope and the trans- mission electron microscope can achieve a much better resolution. However, the samples must be placed in a vacuum chamber, and often coated with metal or other substances. At best those methods resemble a kind of au- topsy. STM and AFM can, in principle, image live biological molecules, and it is possible to image the process.
Because the biological molecules are poor conductors, AFM is more suit- able. The AFM experiments are often performed in air or in an aqueous buffer solution at room temperature.
As long as proper sterilization is carried out, clean environment could be achieved. Since the static-mode AFM often damages the sample, tapping mode is frequently used.
To date, true atomic resolution on biological molecules is rarely achieved. Nevertheless, the nanometer-scale resolution has already provided a lot of unprecedented first-hand microscopic informa- tion on biological molecules.
See Chapter 15 for details. Immobilization and imaging In order to use AFM to take images, the biological molecules must be immo- bilized on an atomically flat surface without impairing its biological activity. In Section 1. Gold surface with a self-assembled monolayer of thiol derivatives is presented. For AFM, an insulating substrate works equally well. One of the best substrates is silanated mica [30].
Mica can be easily cleaved to create atomically flat surfaces. However, biomolecules do not adsorb on native mica surfaces. The amine group -NH2 is exposed and enables the adsorption of biomolecules. The original method was demonstrated on glass surfaces. However, glass surface is not flat enough for nanometer-scale AFM studies. By applying the molecule- combing method to mica, under well-controlled conditions, very straight, parallel DNA strands can be generated, as verified by AFM images [30].
Reproduced from [32], with permission. DNA manipulation The AFM has achieved beyond the target of Feynman: it is capable of not only imaging, but also manipulating the biological molecules. Those DNA strands are first processed with a flowing buffer fluid, to form a more-or-less regular pattern.
Then, the AFM tip is used to manipulate the DNA strands to fabricate designed patterns by the following sequence of actions. The first action is molecular cutting.
By positioning the tip at a pre- determined location of the DNA strand, then pressing it hard, the DNA strand could be broken at a point of nanometer accuracy. The second action is sweeping or pushing. A fragment of a DNA strand could be pushed by the side of the AFM tip, either to move it out of the area of interest, or to reposition it to a predetermined location. In Fig. The tip, which holds the DNA segment, is transferred into a test tube.
Using the polymerase chain reaction process PCR , invented by Kary Mullit [35], the single DNA segment can be multiplied into billions of copies within a few hours in a test tube with the help of an enzyme polymerase. For a rerview of PCR, see Chapter 8 of [36]. Reproduced with permission from [34]. Copyright Institute of Physics. For a review of the Sanger sequencing method, see Chapter 12 of [36]. The proof-of-concept experiment was performed with a relatively small and well-understood DNA molecule, the linearized pRB It has base pairs bp and a theoretical length of nm.
The experiment was performed using Nanoscope IIIa in air at room temperature. The DNA molecule was then dissected at two predetermined points to create an isolated segment, see Fig.
By pushing the side of the tip against that segment, it could adhere onto the tip. By imaging the substrate again with AFM, it appeared the segment was missing, see Fig. The results of PCR were then investigated by Sanger sequencing.
In the very first set of experiments, among 20 attempts, 7 cases were positive: the expected DNA segments were detected [33, 34]. Lecture at an APS meeting. The Archives, California Institute of Technology, see www.
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This means that the vacuum is very good indeed, typically less than 10 Torr that's about 10 times atmospheric pressure! This vacuum is very important, because we do not want rogue oxygen or water molecules sitting on the nice clean sample surface we are trying to study. In order to understand the tunneling current, first we have to talk about "density of states". Electrons in an isolated atom live at specific discrete energy levels.
Likewise in a metal, the electrons must live at specific energy levels, based on the energy landscape of the metal. The difference is, that in a macroscopic piece of metal there are so many electrons that the energy level spacing gets very close together. The levels are so close together that it no longer makes sense to try to list the energy levels of all the electrons.
There are 10 23 electrons in a macroscopic piece of metal, so it would actually take us 10 16 years, that's 6 million times the age of the universe, to write down all the energy levels at a rate of one per second!
Why wouldn't they all just clump together at the lowest point at the bottom of the valley? The answer is that electrons are rather unfriendly characters called fermions. No two fermions are allowed to occupy the same energy state; this is known as the Pauli exclusion principle.
So the electrons must pile on top of each other instead. Electrons are happy sitting in either the tip or the sample, i. But it takes energy to remove an electron into free space. We can think of the vacuum around the tip as an energy hill that the electron would need to climb in order to escape.
In order to bring an electron up and over the vacuum energy barrier from the tip into the sample or vice versa , we would need to supply a very large amount of energy. Climbing hills is hard work! Luckily for us, quantum mechanics tells us that the electron can tunnel right through the barrier.
Note: this only works for particles, not for macroscopic objects.
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