Method of Oligonucleotide purification depends on three key factors:

• Oligonucleotide length
• Application
• Modifications

The first factor is the length of an oligonucleotide. The longer an oligonucleotide sequence is, the more failing sequences present in the crude mixture. Regardless of coupling efficiency, long oligonucleotides of greater than 50 bases are highly recommended to undergo purification. The second factor is application. For example, some oligonucleotides require characterization by ion exchange chromatography to achieve extremely high purity greater than 98%. This is important for cell viability. The preferred method of purification is anion exchange HPLC due to this ability to produce high quality oligos in the cell friendly Na+ salt form. The third factor is modifications. Sometimes a labeled or dual labeled oligo is required for molecular beacon, FRET, biotin-streptavidin, or bio conjugation applications. In these special cases, the hydrophobicity of the modifying group at either the 5’ or 3’ end can be utilized to produce high quality oligos through C18 reverse phase HPLC.

The most common purification techniques used at Biosynthesis are:

• Polyacrylamide gel electrophoresis (PAGE)
• Reverse Phase High Pressure Liquid Chromatography (RP HPLC)
• Anion Exchange High Pressure Liquid Chromatography (AEX HPLC)

Each purification technique has its own strengths and weaknesses. However, if done properly each purification method can produce high quality oligonucleotides.

PAGE:

Polyacrylamide gel electrophoresis is the oldest technique in oligonucleotide purification. Generally, a high percentage acrylamide gel is used to separate the full length products from all failed sequences based on their charge and ability to “run” through the gel matrix. PAGE can be used to separate oligos with lengths greater than 15 bases all the way up to 400 base pairs yielding purities greater than 90%. The advantage of PAGE is that it is the only purification technique that can resolve unmodified oligos of lengths greater than 80 bases. Biosynthesis always recommends long oligonucleotides greater than 80 bases to be purified by PAGE. However, there is one drawback. PAGE purified products will yield a lower final purified product compared to HPLC because it is impossible to recover every bit of full length product from the gel slice. However, the loss in final product is an acceptable tradeoff if high quality, unmodified long oligonucleotides are needed.

Shown below in Figure 1 is an analytical electropharrogram of a 218 base oligonucleotide made and purified by Biosynthesis.

Fig 1.Analytical electropharrogram of a PAGE purified unmodified 269 base oligonucleotide. The band clearly shows that all truncated sequences were removed.

RP HPLC:

Reverse phase is generally the “go-to” standard for HPLC purification of oligonucleotides due its ability for quick mass spec and analytical HPLC quality control of fractions. General knowledge of how RP HPLC works is important to understanding the purification efficiency of RP HPLC. Many find it interesting that RP HPLC actually separates failed oligos from full length products by charge. How is it that a technique based on hydrophobicity of a compound can separate a polar molecule? In reality, RP HPLC of oligonucleotides is actually “ion-pairing chromatography”. This is a technique in which a long-chained alkyl amine is added in low concentration to the mobile phase in order to achieve enhanced resolution. Below is a schematic (scheme. 1) of how an oligonucleotide interacts with in ion-pairing reagent which in turn interacts with the C18 stationary phase of the column.

Scheme 1.Interaction of an oligonucleotide with an ion-pairing reagent at the surface of a C₁₈ support.

This “indirect” interaction is the reason why RP HPLC can only resolveoligos from 8 to 40 bases with greater than 90% purity. However, if greater purity is required, the hydrophobic DMT group of the oligo can be left on the last 5’ base during synthesis, producing greater than 95% pure oligonucleotides. The advantage of both forms of HPLC (RP and AEX) over PAGE is that the yield is typically much higher relative to the starting scale.

A second advantage to reverse phase chromatography is its ability to resolve modified oligos from unmodified oligos. Typically, modifications at the 5’ or 3’ end of an oligonucleotide such as fluorophores, biotin, amino,sulfohydryl, etc. are hydrophobic enough to interact directly with the stationary phase of the C18 column. This leads to highly purified custom modified oligos that can be greater than 95% pure. The two disadvantages to RP HPLC are(unmodified) oligos greater than 40 bases and less than 8 bases cannot be resolved.

It is important to point out that RP HPLC purified oligonucleotides are left in the ion-pairing reagent salt form. Normally, this is the triethylammonium amine (TEAA) salt. If the oligonucleotide is to be used in living systems such cells or animals, TEAA is very toxic. A simple sodium salt exchanged can be performed to remove any TEAA that would cause harm to cells.

Shown below in fig. 2, fig. 3, and fig. 4 is a preparatory chromatogram,mass spectra, andanalytical report, respectively, of a 9mer 3’ Atto 655 modified oligonucleotide that was made and purified by Biosynthesis.

Fig 2.Semi-preparatory RP HPLC chromatogram of a crude 9mer 3’ Atto 655 modified oligonucleotide. The peak at 9.85 minutes represents the unmodified oligonucleotide. The main peak represents the highly pure Atto 655 modified product.

Fig 3.Mass Spectra of main peak collected from preparatory RP HPLC of the 9mer 3’Atto 655 modified oligonucleotide. The calculated molecular weight is 3433.55. The observed molecular weight is 3434.46.

Fig 4.Analytical HPLC report of 9mer 3’ Atto 655 modified oligonucleotide of main peak from preparatory RP HPLC. Fluorophores such as Atto 655 contain enough hydrophobicity to allow for excellent reverse phase separation yielding high purity oligonucleotides.

AEX HPLC:

Anion-exchange chromatography is an established technology with a long history. AEX HPLC is the best form of purification for unmodified oligonucleotides up to 80 bases. The difference between AEX HPLC purification and RP HPLC purification is the way oligonucleotides interact with the stationary phase. In RP HPLC, the interaction was indirect. However, in AEX HPLC the negatively charged phosphate backbone of the oligonucleotide can directly interact with the positively charged stationary phase of the anion exchange column. This results in higher resolution.

There are 3 main advantages to AEX HPLC. The first advantage is that oligomers up to 50 bases can be greater than 95% pure. From 50 to 80 bases, 95% or greater purity can also be achieved, but at lower yields. The second advantage is that AEX HPLC is the only type of purification technique that can successfully separate phosphothiolate, LNA, and BNA modified oligonucleotides. Generally, Biosynthesis recommends AEX HPLC when these modifications are present. However, if a hydrophobic modifying group is present, RP HPLC can also be used. The last advantage is that oligos that elute from an AEX HPLC system are in the Na salt form ready for use in living cells. The only minor setback of AEX HPLC is its slightly higher cost compared to RP HPLC and its lower resolving capability of 3’ or 5’ modifiedoligos. In such cases many customers chose DUAL HPLC purification with both RP and AEX HPLC to achieve 98% or greater purity.

Shown below in Fig. 5, Fig. 6, and Fig. 7 is a preparatory chromatogram, mass spectra, and analytical report respectively of a 28mer oligonucleotide purified by Biosynthesis.

Fig 5.Semi-preparatory AEX HPLC chromatogram of a crude 28mer oligonucleotide. Failure sequences are represented as peaks before the main peak. The small peaks after the main peak are sequences that are not fully de-protected.

Fig 6.Mass Spectra of main peak collected from preparatory AEX HPLC of the 28mer unmodified oligonucleotide. The calculated molecular weight is 8531.58. The observed molecular weight is 8532.17.

Fig. 7Analytical AEX HPLC report of the unmodified 28mer oligonucleotide from the main peak of the preparatory AEX HPLC. Purities exceeding 95% in this case 97.8% are commonly obtained when using AEX HPLC to purify oligonucleotides.

Whatever purification methods our customers decide to choose, Biosynthesis is committed to synthesizing the purest  oligonucleotides.

 The synthetic tetrapeptide Ac-SDKP, also called seraspenide or goralatide, has the chemical structure 1-(N2-(N-(N-acetyl-L-seryl)-L-alpha-aspartyl)-L-lysyl)-L-proline, the molecular formula C20H33N5O9, and a molecular weight of 487.50412 dalton. This tetrapeptide is involved in angiogenesis and the regulation of the immune response by blocking the proliferation of lymphocytes. It is generated by the cleavage of thymosine beta4 and inhibits the proliferation of hematopoietic stem cells and the proliferation and secretion of fibroblasts in the myocardium and the glomeruli. Lymphocytes are a type of white blood cells in the vertebrate immune system and are part of the adaptive immune system. The seraspenide peptide inhibits cell cycle entry of normal hematopoietic stem cells. Ac-SDKP protects hemopoiesis, the blood cell formation from established blood cell precursors, against damage caused by cytarabine, cyclophosphamide and carboplatin in mice. Hematopoietic stem cells give rise to all other blood cells. The renin–angiotensin system, a hormonal system that regulates blood pressure and water or fluid balance in mammals, is central to regulation of blood pressure and electrolyte homeostasis. N-Ac-Ser-Asp-Lys-Pro is an inhibitor of primitive hematopoietic cells and has been reported to block the proliferation of clonogenic cells freshly isolated from normal marrow and cord blood. Ac-SDKP can cause a transient decrease in nonadherent cell and progenitor output when added repeatedly to normal long-term marrow cultures. Ac-SDKP is detectable in experiments with marrow from patients with chronic myeloid leukemia. Clonogenic cells are cells that have the potential to proliferate and to give rise to a colony of cells. Only some daughter cells from each generation retain the potential to proliferate. The angiotensin-converting enzyme, a key protease, has a range of substrates, including N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP). The peptide Ac-SDKP is cleared almost exclusively by the angiotensin-converting enzyme. N-Acetyl-Ser-Asp-Lys-Pro is a negative regulator of haematopoietic stem cell differentiation and a potent antifibrotic agent. AZT (3'-azido-3'-deoxythymidine), the main antiviral drug used in AIDS treatment, is toxic to hematopoietic progenitor cells and is known to induce anemia, a decrease in number of red blood cells, and neutropenia, the abnormally low number of neutrophils or white blood cells. The tetrapeptide AcSerAspLysPro (AcSDKP, Seraspenide) has been shown to increase the survival of mice subjected to high doses of chemotherapy and to reversibly block the cycling of human granulocyte-macrophage colony forming unit and burst forming unit erythroid progenitors. Furthermore, it has been suggested that AcSDKP may be an efficient factor in preserving progenitors against AZT-induced hematopoietic toxicity. Research indicates that the peptide could protect hematopoietic stem cells during anti-neoplastic treatments leaving cancerous cells unprotected. The tetrapeptide opposes the effects of TGFbeta and could prevent fibrosis after myocardial infarcts and glomeruli fibrosis during the natural course of diabetic nephropathy.

Neuropeptides for Neuroscience Research

One of the greatest challenges in neuroscience research is to understand how biologically active molecules such as small molecules and peptides function at different levels, starting at the atomic level to the ensembles of neuronal networks. Advances made in chemical and synthetic biology over the last few decades have given researchers new insights into bioactive molecular actions. One important key feature that has emerged in recent years is the observation that adult mammalian brains exhibit much more plasticity and regenerative capacity than previously thought. During neurogenesis, the process by which neurons are generated from neural stem and progenitor cells, adult brains are able to generate functionally integrated new neurons. Progenitor cells are ancestor cells that can differentiate into specific cell types. Neuroscientist are just now beginning to study the basic properties of adult neural stem cells and the molecular, cellular and circuitry mechanisms that regulate the sequential adult neurogenesis process in vivo. These studies include the new field called “neuroepigenetics” that investigates the function of novel active DNA modifications and their physiological role in the nervous system.

The discovery of neuropeptides in the mammalian nervous system started a revolution in traditional physiology. Groundbreaking research in physiology, endocrinology, and biochemistry during the last century has let to this discovery. The three notions governing peptide research in the early years of peptide based neuroscience are: (1) peptide hormones are chemical signals in the endocrine system; (2) neurosecretion of peptides is a general principle in the nervous system; and (3) the nervous system is responsive to peptide signals. These ideas have contributed to how neuropeptides are defined today: “Neuropeptides are small substances similar to proteins produced and released by neurons through the regulated secretory route that act on neural substrates.” The beginnings of neuropeptide research can be traced back to the histories of physiology, pharmacology and biochemistry.

Originally the term “neuropeptide” indicated a small protein molecule present in neurons. However, in the late 1970s and the 1980s of the last century, many neuropeptides were localized by immunocyto-chemistry to discrete cell populations of the central and peripheral nervous system leading to the concept of “chemical neuroanatomy” as part of neurobiology. However, recently the field of neuropeptide research or biology has dramatically expanded and today the research goals involve the development of pharmacologically active compounds that are capable of crossing the blood–brain barrier to exert their biological function(s) in vivo and in the construction of genetic vectors useful for gene therapy.

How are neuropeptides defined today?

The term ‘neuropeptides’ was first introduced by D. de Wied in 1971 to describe fragments of peptide hormones lacking the activity of the intact hormone molecule but that were able to produce behavioral changes. Subsequently peptide hormones, their fragments, endogenous opioid or morphine-like peptides as well as a large number of other biogenic peptides became classified as neuropeptides. All these peptides are classified based on a number of common features including their origin, biosynthesis, secretion, metabolism, and effectiveness. Neuropeptides are found in the nervous tissue, in peptide-secreting cells present in various organs such as the gut, placenta, heart, lungs, the immune system and other tissues, and are active at extremely low concentrations. The more modern definition of a neuropeptide is as follows: “A neuropeptide is a peptide found in the nervous system which has biological effects when injected.”

Neuropeptides have now been found to be the most diverse class of signaling molecules in the brain that participate in many physiological functions. Almost 70 genes have been identified in the mammalian genome to encode for neuropeptide precursors and a multitude of bioactive neuropeptides. Unfortunately the boundary between a neuropeptide and a neuroprotein can be blurry. This is exemplified in the findings that there are several subfamilies of peptides and small proteins which display most of the hallmarks of neuropeptides. For example among cytokines, peptide hormones, and growth factors there are several subfamilies of peptides that perform similar functions as neuropeptides such as neural chemokines, cerebellins, neurexophilins, and granins. The "cytokine" family alone includes a large and diverse family of regulators produced throughout the body by cells of diverse embryological origin. The use of in situ hybridization and immunocytochemistry allowed scientists to unambiguously demonstrate for many neuropeptides that the transcript and peptide products are produced by neurons. For a few neuropeptides the biosynthesis of the peptides could be demonstrated by the incorporation of radioactive amino acids using pulse-chase experiments.

Why are neuropeptides important?

The recent Annual Neuroscience Meeting held in San Diego from November 9 to 13 demonstrated the importance of all classical neuropeptides as well as of new putative neuropeptides for brain research. The meeting was attended by more than 30,000 neuroscientists and students and the forest of presented posters was so huge that one was only able to visit a selection of them during the meeting hours. One particular peptide that stood out of the crowd is the NAP peptide. This peptide offers new hope to find a cure to prevent the onset of Alzheimers disease or any similar disease, and, hopefully, a few other neuro-degenerative diseases that involve protein unfolding or misfolding events in the mammalian and human brain.

Neuropeptides can be neuroprotective!

“NAP peptide (Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln) is a neuroprotective peptide that shows cognitive protection in patients with amnestic mild cognitive impairment, a precursor to Alzheimer's disease. NAP exhibits potent neuroprotective properties in several in vivo and cellular models of neural injury. While it has been found in many studies to affect microtubule assembly and/or stability in neuronal and glial cells at fM concentrations, it has remained unclear whether it acts directly or indirectly on tubulin or microtubules. We analyzed the effects of NAP (1 fM-1 µM) on the assembly of reconstituted bovine brain microtubules in vitro and found that it did not significantly (p < 0.05) alter polymerization of either purified tubulin or of a mixture of tubulin and unfractionated microtubule-associated proteins. NAP also had no significant effect (p < 0.05) on the growing and shortening dynamics of steady-state microtubules at their plus ends, nor did it alter the polymerization or dynamics of microtubules assembled in the presence of 3-repeat or 4-repeat tau. Thus, the neuroprotective activity of NAP does not appear to involve a direct action on the polymerization or dynamics of purified tubulin or microtubules.” [Source: Mythili Yenjerla, Nichole E LaPointe, Manu Lopus, Corey Cox, Mary Ann Jordan, Stuart C Feinstein, and Leslie Wilson; The Neuroprotective Peptide NAP Does Not Directly Affect Polymerization or Dynamics of Reconstituted Neural Microtubules. J Alzheimers Dis. 2010 January 1; 19(4): 1377–1386. doi: 10.3233/JAD-2010-1335. PMCID: PMC2844470, NIHMSID: NIHMS179597.]

Gene expression, biosynthesis and regulated release in neurons

Neurons use neuropeptides for signaling to other cells. Expression of the genes coding for neuropeptides and their biosynthesis occur in neurons. This has been shown using in situ hybridization and immunocytochemistry. Usually only peptides produced in the nervous system by neurons are called neuropeptides. However, astrocytes and glial cells apparently can also have a regulated secretory pathway and therefore, putative neuropeptides may be recognized in peptide families expressed by glial cells.

Recent research suggest that many neurotransmitters, neuropeptides and calcium binding proteins appear early during development of the cerebellum, have specific temporal and spatial expression patterns, may have functions other than those found in the mature neural systems, and may be able to interact with each other during early development. The basis for regulation of chemical communication is controlled secretion of the signal molecules involved. Neurons expressing classical neuropeptides use the regulated secretory pathway. In this pathway biosynthesized peptides are stored in large dense-cored vesicles and released in a controlled manner upon a stimulus. A signal peptide sequence is needed for entry of the newly synthesized gene product into the lumen of the endoplasmic reticulum (ER) as part of the secretion routes. Usually the signal peptide is short, containing 20 to 25 amino acids at the N-terminal end of the precursor, called the prepro-peptide. This signal peptide is removed from the nascent precursor during protein synthesis when it passes through the ER membrane, leaving the pro-neuropeptide in the ER-Golgi for further sorting into the regulated secretory pathway. Next, neuropeptide precursors are sorted in the trans-Golgi network into the vesicles of the secretory pathway. The resulting protein content is acidified and condensed in the vesicles, and activated proteolytic enzymes process the precursor protein into neuropeptides. Pairs of the basic amino acids lysine (Lys, K) and arginine (Arg, R), or, sometimes, a single Arg in the appropriate structural environment of the precursor, serve as recognition sites and substrates of prohormone convertases (PCs). Next, differential cleavage of the neuropeptide precursor by the proteases leads to the generation of specific peptides and cell-specific different sets of peptides from the same precursor. Peptides generated in this way can be subject to further modification by peptidyl-aminotransferase (PAM). This enzyme uses a C-terminal glycine (Gly, G) as amide donor for the preceding amino acid which results in a peptide with an amidated C-terminus. This amide group has been found to occur in many neuropeptides and is often essential for its biological activity. Furthermore, many neuroppetides have been found to contain post-translational modifications including N- and O-glycosylation, phosphorylation, sulfation, and acetylation. These are found on stored and secreted peptides and contribute to their biological properties. The resulting neuropeptides are stored in mature granules or dense-cored vesicles where they await release upon a stimulus.

Neuropeptides can function as hormones

Some neuropeptides can also function as hormones. A hormone is defined as a substance secreted by one organ and acting on another after transport by the bloodstream. This allows cells at different locations of an organism to communicate in an endocrine fashion. Since many peptide hormones are also synthesized by neurons these peptides therefore belong to the class of neuropeptides. However, many classical neuropeptides are also synthesized by endocrine glands and function peripherally as hormones which can make the classification of these peptides somewhat confusing. By definition hormones are excluded as being neuropeptides if they are not synthesized by neurons, even if they signal to the brain. Leptin and insulin are examples. In addition, sometimes the relationships between the nervous system and peripheral endocrine glands are complicated by alternative processing and splicing. Hormones are defined as being chemical substances formed or synthesized in one or part of an organ in the body and are carried in the blood to another organ or part. Depending on the specificity or effect of the hormone the targeted organ may be altered in its functional activity or even in its structure by the effect of the hormone. As new peptides are discovered in various tissues that can function as neuropeptides the current classification schemes may need to be expanded.

How to design peptides useful for immunization using multiple antigen peptides (MAPs)

The design of peptides to generate antibodies against protein epitopes is still considered an art. However, experienced researchers can use an educated approach for the design of immune response inducing peptide sequences for example by determining the amino acid sequence regions exposed to the surface of a protein. One approach is to review the folding of proteins and to search for β-turn loops where the amino acid residues at the turns are pointed to the surface of the proteins. This approach works well for proteins for which the 3D structures are available in a public data base such as the Pubmed Structure database [http://www.ncbi.nlm.nih.gov/structure/] or the RCSB PDB database [http://www.rcsb.org/pdb/home/home.do]. Another approach is to screen the protein sequences for sequence regions that have a high probability to induce immunization using bioinformatic methods. The following web link can help to achieve this:

The multiple antigen peptides (MAP) system represents another approach to anti-peptide antibody elicitation. It has been reported that MAPs can be used for the production of high-titer anti-peptide antibodies as well as synthetic peptide vaccines. The system was first described by Dr. James Tam. A MAP contains a small immunologically inert core molecule of radially branched lysines dendrites onto which a number of peptide antigens are anchored. The result is a large macromolecule which has a high molar ratio of peptide antigen to core molecules and does not require further conjugation to a carrier protein. The α- and ε-amino functional groups of lysine residues are used to form a dendrimeric type core to which multiple peptide chains are attached. Depending on the number of lysines used to synthesize the core structure different numbers of peptide branches can be synthetically attached.

1. What is the optimal length of the peptide? I recently read a paper that indicated 30 mer peptides perform better than 20 mer peptides in an ELISA based assay.
   • We have used many 20mer peptides for ELISA assays and have found no problem in their use. All have worked very well. However, in general we agree that longer peptides will bind stronger to their targets than shorter peptides if using ELISA plates.
   • The optimal length of peptides used for antibody production can vary from 15 to 22 amino acids. Class I peptides that are held within the MHC groove are usually 8 to 9 amino acid residues long. However, class II peptides are between 8 and 30 amino acid residues long.
2. Do the peptides need to be conjugated to a carrier protein and if so what carrier is best? My concern here is steric hindrance if peptides themselves are conjugated to the beads. Therefore mentioned paper suggested that BSA was better than KLH, as KLH caused higher non-specific reactivity.
   • It is not necessary to conjugate peptide to carrier protein for assay development. If one has to be used, BSA or OVA are good carrier proteins that can be used for assay development. If a researcher wants to use carboxylated polystyrene Luminex beads for conjugation, one important point is that the conjugation condition for the carrier protein-peptide conjugate has to be optimized. The reason for this is that after the conjugation reaction many amine groups on the carrier proteins are occupied by peptides. This fact will make it difficult to allow the conjugation to the beads as well. The solution to this problem could be the use of different beads that have different functional groups or to control the conjugation degree of the peptide to the carrier protein. Another method to avoid carrier protein conjugation is to consider adding a spacer (such as a PEG spacer) to the N-terminus of peptides that contains a terminal amine group. This can be done by using a peptide synthesis approach to conjugate peptides to the beads. This method works very well and the peptides can be further conjugated to the carboxylated polystyrene Luminex beads.
3. If MAPs are used are the primary amines on lysines still available for conjugation to microplex beads?
   • In the case of MAPs, the lysines located in the MAP core will not be available for conjugation. The reason for this is that the peptide chains are branched from the alpha and epsilon amine groups of the lysines. The only available groups will be the –COOH group of the MAP and the N-terminus of the 4 or 8 branches of the peptide. In this case, instead of carboxylated polystyrene Luminex beads, different beads with different functional groups will need to be used. The figure below illustrates the structure of a MAP:
     Structure of a MAP.
4. What purity of the peptides should be used?
   • Both, the map core and the selected peptide are usually synthesized on a solid support. When cleaved off, the released MAPs usually exits as heterogeneous molecule with a purity of >85% which can be used for antibody production without further purification.

A brief history of firsts in nanotechnology: Nanotechnology started in 1959 when Feynman gave an after-dinner talk describing molecular machines built with atomic precision. In 1974, Taniguchi used the term "nano-technology" in a paper on ion-sputter machining. The molecular nanotechnology concept was coined by Drexler at MIT in 1977. The first technical paper on molecular engineering to manufacture with atomic precision was published in 1981, when the scanning tunneling microscope (STM) was invented. The Buckyball was discovered in 1985, and the first book on nanotechnology was published in 1986. During the same year, atomic force microscopy (AFM) was invented, and the first nanotechnology organization was formed. The first protein was engineered in 1987, and many firsts followed in nanotechnology, including the publication of the first textbook about the field in 1992, as well as the first nanomedicine book published in 1999. And it all starts with particles.

Let us find out what a particle is. A particle is a small, localized object that behaves as a whole unit and can be described using physical properties such as volume, size and mass. A nano-particle is "a particle having one or more dimensions of the order of 100 nm or less." Usually, fine particles range in their size between 2,500 and 100 nanometers. However, ultrafine particles, or nanoparticles, range in sizes between 100 and 1 nanometers. During the 1970-80’s, when the first thorough fundamental studies were running with "nanoparticles" in the United States (by Granqvist and Buhrman) and Japan, (within an ERATO Project), they were called "ultrafine particles"(UFP). During the 1990s, before the National Nanotechnology Initiative was launched in the United States, the new name, "nanoparticle" had become fashionable. Nanoclusters have at least one dimension between 1 and 10 nanometers and a narrow size distribution. Nanopowders are agglomerates of ultrafine particles, nanoparticles, or nanoclusters. Nanometer-sized single crystals or single-domain ultrafine particles, are often referred to as nanocrystals. The field of nanotechnology is rapidly developing, and new types of nanomaterials are being developed constantly. It is expected that nanomaterials will be developed at several levels, as part of material devices and systems. Many nanomaterials are now commercially available. The cells of living organisms are typically 10 μm in size. Parts of the cells are even much smaller, in the sub-micron size range.

Figure 1: This figure illustrates the size of a nanoparticle. If a nanoparticle was the size of a football - this image shows how atoms, cells and organisms would compare at a more familiar scale to humans.

Proteins that make up the cells nanomachinery are just around 5 nanometers (nm) in size. These are the sizes of the smallest man-made nanoparticles. Their size allows them to be used as probes to study the cells’ biological processes. Typically, for biological applications, nanomaterials are selected for their optical and magnetic properties. However, nanomaterials are also applied for novel electronic, optoelectronis, and memory devices. Figure 1 illustrates the size of nanoparticles.

The fact that nanoparticles exist in the same size domain as proteins allows them to be used to label or tag proteins, either in vivo or in vitro. To allow the interaction of nanoparticles with the biological target, a molecular coating or layer acting as an interface will need to be attached to the particle. Examples of biological coatings include antibodies, biopolymers such as collagen, or monolayers of small biocompatible molecules. Since the use of optical detection techniques are widely used in biological research, nanoparticles should either fluoresce or change their optical properties in response to a biological function. Figure 2 illustrates the sizes and shapes of a few nanoparticles. A list of applications involving nanoparticles follows below.

Figure 2: Sizes and shape of different nanoparticles. This cartoon shows the morphology of nanoparticles that can be used for neuroimaging. Source: Moresco & Masserine 2012.

Some applications of nano-materials in biology and medicine are:

Fluorescent biological labels for fluorescent signaling
Drug and gene delivery
Bio detection of pathogens
Detection of proteins – Antigen detection
Probing of DNA structure
Tissue engineering
Tumor destruction via heating (hyperthermia)
Separation and purification of biological molecules and cells
Linker activated nanoparticles
Biocompatible nanoparticles
MRI contrast enhancement
Molecular imaging such as computed tomography (CT), positron emission computed tomography (PET), single photon emission computed tomography (SPECT), and magnetic resonance (MRI),
Phagokinetic studies

Figure 3: Representation of multifunctional iron oxide nanoparticles showing multiple modes of functionalization.

Figure 4: Representation of multifunctional nanoparticles.

Nanoparticles that usually form the core of nano-biomaterials can be composed of inorganic, polymeric materials or can be in the form of nano-vesicles surrounded by a membrane or a layer. The shapes of these particles can come in different morphologies. Furthermore, nanoparticles can be functionalized in multiple ways. Figures 3 and 4 show the graphical representation of different ways to functionalize nanoparticles. For example, as depicted in figure 3, the particles can be conjugated to different types of molecules, such as antibodies, green fluorescent protein (GFP), avidin, or streptavidin, as well as to DNA/RNA oligomers and gold particles or, as shown in Figure 4, contrast agents useful for CT/MRI imaging, radiotracers useful for PET/SPECT imaging, functionalized with drugs for targeted drug delivery or specific ligands, as well as special surfaces such as hydrophilic surfactants to enhance biocompatibility.

It is thought that nanoparticles will play an increasing role in nanomedicine in the future. Nanomedicine applies nanotechnology with the goal to improve the quality of human lives. Useful medical applications of nanoparticles include improved drug delivery, such as protein, peptide, and oligonucleotide delivery in biological systems, nanoparticles to specific targets in tumors and cancers, and nanoparticles for tissue visualization to enhance surgical techniques or to visualize tumors. It is hoped that it will become possible in the near future to design nanorobots or nanomachines that allow for the repair of damage parts of the cell.

More recently, many companies have begun to use nanotechnologies. The majority of the companies are small recent spinouts of various research institutions. Most of the companies are developing applications for the pharmaceutical industry, mainly to enhance or enable drug delivery. Other companies exploit quantum size effects in semiconductor nanocrystals to tag biomolecules or use gold nanoparticles for the labeling of various cellular parts. Cytodiagnostics, one such company, provides gold, silver, and magnetic nanoparticles with sizes ranging from 5 nm to 400 nm. These particles can be conjugated to biomolecules such as antibodies, BSA, KLH, and many others.

Biosynthesis Inc. in Lewisville, Texas offers custom conjugation of antibodies or other proteins to this type of nanoparticles. To be able to perform custom conjugations, approximately 1-2 mg of purified and lyophilized antibody (or any other protein) is required. 

Services include: 1. antibody or protein sourcing, if needed; 2. conjugation of a customer protein to selected gold nanoparticles; and 3. Purification of the conjugate.

Furthermore, oligonucleotides and other molecules can be conjugated to gold nanoparticles as well. Biosynthesis provides custom conjugation of single-stranded or double-stranded oligonucleotides to gold nanoparticles with sizes ranging from 5 nm to 200 nm. Final conjugate yield depends on both sizes of the oligo and nanoparticle. For a 20 mer oligo, the loading would be 0.2-1 OD/mg nanoparticle. The smaller the particle size, the higher yield.

Services include: 1. synthesis of oligonucleotides for conjugation. The customers usually supplies the nucleotide sequence, and decides which terminal (5' or 3') will be attached to the gold surface, and Biosynthesis Inc. does the rest; 2. Conjugation of the oligonucleotide to a gold nanoparticle size of customer’s choice; 3. Purification of the conjugate.

The following table shows a list of nanomedical technologies.

Cytodiagnostics, another company, offers a unique proprietary protocol that produces particles with uniform shapes and a narrow size distribution. The gold nanoparticle surface can be modified to allow for the conjugation to molecules such as biotin and other molecules of choice. Furthermore, particles can be functionalized with carboxyl, amine, and methyl groups, among others.

Silver Nanoparticles are also available with core sizes of 40 nm - 100nm

High-quality monodisperse silver nanoparticles with a narrow size distribution (CV <15%) are available as well. Nanosilver products are ideal for a wide array of applications such as “Conjugate Development,”, “Sensor Development,”, “Molecular Imaging’,’Surface Enhance Raman Spectroscopy (SERS),”, and“Bactericidal applications.”

Superparamagnetic iron nanoparticles, both in the metallic, and oxide forms, can be used for bioconjugation as well and are widely used in the life sciences. Typical applications include but are not limited to the separation and purification of biomolecules, such as proteins and DNA, from complex mixtures, as well as in immunoprecipitation protocols. By using only a magnet for fast pull-down and isolation of magnetic particles, cumbersome and long purification protocols using columns or centrifugation are not needed. Other applications may include medical diagnostics, catalysis, probing agents to indirectly study the structure of mixed self-assembled monolayers (SAMs), solid phase extraction, anode materials for Li-ion batteries, and electromagnetic interference (EMI) shielding materials. Source: http://www.cytodiagnostics.com/magnetic_nanoparticles.php

The company Nanoprobes offers 1.4 nm Nanogold® particles. These are gold compounds that are not just adsorbed to proteins, like colloidal gold, but covalently reacts at specific sites under mild buffer conditions with molecules that are selected for conjugation. A well-defined product can be synthesized that can be purified chromatographically.

 Source: http://www.nanoprobes.com/products/LabRgts.html#feat

Features of Nanogold® Unparalleled penetration of conjugates up to 40 µm. Higher density of immunolabeling than with larger gold probes. Can be conjugated to any molecule with a suitable reactive group. Available with different reactivities. Extremely uniform 1.4 nm gold particle Label at specific sites, which do not obstruct native reactivity. Close to stoichiometric labeling. Reacts under mild, neutral conditions Conjugates are easily isolated by gel filtration. Conjugates are stable to a wide range of pH and ionic strengths. High Representation of multifunctional ironstability: conjugates show unchanged reactivity after storage for a year.