# Nanoinjection
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'''Nanoinjection''' is the process of using a microscopic lance (i.e. a nanoneedle) and [electrical forces](/source/Electrostatics) to deliver [DNA](/source/DNA) to a cell. It is claimed to be more effective than [microinjection](/source/microinjection) because the lance used is ten times smaller than a [micropipette](/source/micropipette) and the method uses no fluid. The nanoinjector mechanism is operated while submerged in a [pH](/source/pH) buffered solution. Then, a positive electrical charge is applied to the lance, which accumulates negatively charged DNA on its surface. The nanoinjector mechanism then penetrates the [zygotic](/source/Zygote) membranes, and a negative charge is applied to the lance, releasing the accumulated DNA within the cell. The lance is required to maintain a constant elevation on both entry and exit of the cell.[{{Cite journal|title=A self-reconfiguring metamorphic nanoinjector for injection into mouse zygotes|journal=Review of Scientific Instruments|volume=85|issue=5|page=055005|doi=10.1063/1.4872077|pmid=24880406|year = 2014|last1 = Aten|first1 = Quentin T.|last2=Jensen|first2=Brian D.|last3=Burnett|first3=Sandra H.|last4=Howell|first4=Larry L.|bibcode=2014RScI...85e5005A }}]
Nanoinjection results in a long-term cell viability of 92% following the [electrophoretic](/source/Electrophoresis) injection process with a 100 nm diameter [nanopipette](/source/nanopipette), the typical diameter of nanoinjection pipet.[{{Cite journal|last1=Simonis|first1=Matthias|last2=Hübner|first2=Wolfgang|last3=Wilking|first3=Alice|last4=Huser|first4=Thomas|last5=Hennig|first5=Simon|date=2017-01-25|title=Survival rate of eukaryotic cells following electrophoretic nanoinjection|journal=Scientific Reports|language=En|volume=7|article-number=41277|doi=10.1038/srep41277|pmid=28120926|pmc=5264641|bibcode=2017NatSR...741277S |issn=2045-2322}}]
Single cell transfections are used to virtually transfer any type of [mammal](/source/mammal)ian cell into another using a syringe which creates an entry for DNA to be released. A nano needle is used as a mechanical vector for [plasmid](/source/plasmid) DNA. The method can be improved further with [Atomic Force Microscopy](/source/Atomic_force_microscopy) or AFM. In order to avoid causing permanent damage to the cell or provoke cellular leaking of intracellular fluid, AFM is a tool of choice, as it allows for precise positioning of the DNA, allowing for tip penetration into the [cytosol](/source/cytosol), which is critical for viable DNA transfer into the cell.[{{Cite journal|date=2007-04-13|title=Single cell transfection using plasmid decorated AFM probes|journal=Biochemical and Biophysical Research Communications|language=en|volume=355|issue=3|pages=632–636|doi=10.1016/j.bbrc.2007.01.190|pmid=17316557|issn=0006-291X|last1=Cuerrier|first1=Charles M.|last2=Lebel|first2=Réjean|last3=Grandbois|first3=Michel |bibcode=2007BBRC..355..632C }}]
Reasons to use nanoinjection include the insertion of genetic material into the [genome](/source/genome) of a zygote. This method is a critical step in understanding and developing gene functions.
Nanoinjection is also used to genetically modify animals to aid in the research of [cancer](/source/cancer), [Alzheimer's](/source/Alzheimer's_disease) disease, and diabetes.[{{Cite journal |last1=Peter Rosenfeld |first1=J. |last2=Hou Huang |first2=Kun |last3=Yan Xia |first3=Lang |date=1990-02-05 |title=Effects of single and simultaneous combined nanoinjections of Met-enkephalin into rat midbrain and medulla on activity of differentially nociresponsive ventral medullary neurons |url=https://dx.doi.org/10.1016/0006-8993%2890%2990397-T |journal=Brain Research |volume=508 |issue=2 |pages=199–209 |doi=10.1016/0006-8993(90)90397-T |pmid=2306611 |issn=0006-8993|url-access=subscription }}] Many papers in the 1990s used modified microinjection techniques referred to as "nanoinjections" but do not match the modern definition of nanoinjection.[{{Cite journal |last1=Åkerman |first1=Gun |last2=Balk |first2=Lennart |date=1995-01-01 |title=A reliable and improved methodology to expose fish in the early embryonic stage |url=https://dx.doi.org/10.1016/0141-1136%2894%2900010-M |journal=Marine Environmental Research |series=Responses of Marine Organisms to Pollutants |volume=39 |issue=1 |pages=155–158 |doi=10.1016/0141-1136(94)00010-M |bibcode=1995MarER..39..155A |issn=0141-1136|url-access=subscription }}][{{Cite journal |last1=Ishaq |first1=Rasha |last2=Åkerman |first2=Gun |last3=Näf |first3=Carina |last4=Balk |first4=Lennart |last5=Bandh |first5=Cecilia |last6=Broman |first6=Dag |date=1999-03-01 |title=Organic pollutant characterization and toxicity testing of settling particulate matter by nanoinjection in sea trout (Salmo trutta) eggs |url=https://dx.doi.org/10.1002/etc.5620180323 |journal=Environmental Toxicology and Chemistry |language=en |volume=18 |issue=3 |pages=533–543 |doi=10.1002/etc.5620180323 |bibcode=1999EnvTC..18..533I |issn=0730-7268|url-access=subscription }}][{{Cite journal |last1=Villalobos |first1=Sergio A. |last2=Papoulias |first2=Diana M. |last3=Meadows |first3=John |last4=Blankenship |first4=Alan L. |last5=Pastva |first5=Stephanie D. |last6=Kannan |first6=Kurunthachalam |last7=Hinton |first7=David E. |last8=Tillitt |first8=Donald E. |last9=Giesy |first9=John P. |date=2000-02-01 |title=Toxic responses of medaka, D-rR strain, to polychlorinatednaphthalene mixtures after embryonic exposure by in ovo nanoinjection: A partial life-cycle assessment |url=https://dx.doi.org/10.1002/etc.5620190224 |journal=Environmental Toxicology and Chemistry |language=en |volume=19 |issue=2 |pages=432–440 |doi=10.1002/etc.5620190224 |bibcode=2000EnvTC..19..432V |issn=0730-7268|url-access=subscription }}][{{Cite journal |last1=Vasjari |first1=M. |last2=Mirsky |first2=V. M. |date=2000-11-01 |title=Calibrated nanoinjections of mercury vapor |url=https://doi.org/10.1007/s002160000555 |journal=Fresenius' Journal of Analytical Chemistry |language=en |volume=368 |issue=7 |pages=727–729 |doi=10.1007/s002160000555 |pmid=11227555 |issn=1432-1130|url-access=subscription }}][{{Cite journal |last1=Wright |first1=Peggy J. |last2=Tillitt |first2=Donald E. |date=1999-11-01 |title=Embryotoxicity of Great Lakes lake trout extracts to developing rainbow trout |url=https://www.sciencedirect.com/science/article/pii/S0166445X99000107 |journal=Aquatic Toxicology |volume=47 |issue=2 |pages=77–92 |doi=10.1016/S0166-445X(99)00010-7 |bibcode=1999AqTox..47...77W |issn=0166-445X|url-access=subscription }}]
* '''2004''' - A nanoscale surgery on a living cell is performed with a sharpened Atomic Force Microscope (AFM) tip. Through sharpening an AFM tip to 200-300 nm in diameter, they found they could successfully perforate both the cellular and nuclear envelopes then withdraw the tip with minimal damage.[{{Cite journal |last1=Obataya |first1=Ikuo |last2=Nakamura |first2=Chikashi |last3=Han |last4=Nakamura |first4=Noriyuki |last5=Miyake |first5=Jun |date=2005-01-01 |title=Nanoscale Operation of a Living Cell Using an Atomic Force Microscope with a Nanoneedle |url=https://doi.org/10.1021/nl0485399 |journal=Nano Letters |volume=5 |issue=1 |pages=27–30 |doi=10.1021/nl0485399 |pmid=15792407 |bibcode=2005NanoL...5...27O |issn=1530-6984|url-access=subscription }}]
* '''April, 2007''' - The first usage of a nanoinjection format similar to modern day nanoinjection is published. They used a custom AFM tip coated in an [EGFP](/source/EGFP) encoding plasmid to transfect individual [eukaryote](/source/eukaryote) cells. They found that they could easily observe when the tip punctured the membrane from the force readings, but could not control how much was released. They successfully caused minimal disturbance to the cells, but only 30% of cells were successfully transfected.[{{Cite journal |last1=Cuerrier |first1=Charles M. |last2=Lebel |first2=Réjean |last3=Grandbois |first3=Michel |date=2007-04-13 |title=Single cell transfection using plasmid decorated AFM probes |url=https://www.sciencedirect.com/science/article/pii/S0006291X0700246X |journal=Biochemical and Biophysical Research Communications |volume=355 |issue=3 |pages=632–636 |doi=10.1016/j.bbrc.2007.01.190 |pmid=17316557 |bibcode=2007BBRC..355..632C |issn=0006-291X|url-access=subscription }}]
* '''May, 2007''' - A lab at the University of California publishes their findings with using [carbon nanotubes](/source/Carbon_nanotube) as the lance in the AFM. They attached [quantum dots](/source/Quantum_dot) to the outside of the lance via a [disulfide bridge](/source/disulfide_bridge). This method allowed for high precision and undetectable membrane damage, but is slow and can only transfer limited types of payloads. It requires 15-30 minutes to release the payload in the cell and required a payload that could form disulfide bridges.[{{Cite journal |last1=Chen |first1=Xing |last2=Kis |first2=Andras |last3=Zettl |first3=A. |last4=Bertozzi |first4=Carolyn R. |date=2007-05-15 |title=A cell nanoinjector based on carbon nanotubes |journal=Proceedings of the National Academy of Sciences |volume=104 |issue=20 |pages=8218–8222 |doi=10.1073/pnas.0700567104 |doi-access=free |pmc=1895932 |pmid=17485677 |bibcode=2007PNAS..104.8218C }}]alt=Positively Charged Nanoinjection Needle Attracts Negatively Charged DNA for Subsequent Injection|thumb|237x237px|Positively Charged Nanoinjection Needle Attracts Negatively Charged DNA for Subsequent Injection
* '''2011''' - A team at BYU successfully attached and released DNA from the outisde of a machined silicone lance. They found that through introducing a positive charge to the lance the negatively charged DNA could be attached until the polarity was flipped. In this way over 6,000 DNA molecules could be delivered with minimal damage to the membrane or cell.[{{Cite journal |last1=Aten |first1=Quentin T. |last2=Jensen |first2=Brian D. |last3=Burnett |first3=Sandra H. |last4=Howell |first4=Larry L. |date=December 2011 |title=Electrostatic Accumulation and Release of DNA Using a Micromachined Lance |journal=Journal of Microelectromechanical Systems |volume=20 |issue=6 |pages=1449–1461 |doi=10.1109/JMEMS.2011.2167658 |bibcode=2011JMemS..20.1449A |issn=1941-0158}}]
* '''May 2014''' - The same team at BYU used their nanoinjection system to inject 3000 [mouse](/source/mouse) embryos and found that it successfully delivered the DNA payload while minimizing cell death rates. They found a low increased death rate of roughly 7.7% more in the injected zygotes compared to the control.[{{Cite journal |last1=Aten |first1=Quentin T. |last2=Jensen |first2=Brian D. |last3=Burnett |first3=Sandra H. |last4=Howell |first4=Larry L. |date=December 2011 |title=Electrostatic Accumulation and Release of DNA Using a Micromachined Lance |journal=Journal of Microelectromechanical Systems |volume=20 |issue=6 |pages=1449–1461 |doi=10.1109/JMEMS.2011.2167658 |bibcode=2011JMemS..20.1449A |issn=1941-0158}}]
* '''May 2014''' - A team of researchers constructs an array of solid silicone microlances to inject tens of thousands of cells simultaneously. They found successful delivery to as many as 78% of the cells in the sample and a 78-91% survival rate.[{{Cite journal |last1=Lindstrom |first1=Zachary K. |last2=Brewer |first2=Steven J. |last3=Ferguson |first3=Melanie A. |last4=Burnett |first4=Sandra H. |last5=Jensen |first5=Brian D. |date=2014-05-01 |title=Injection of Propidium Iodide into HeLa Cells Using a Silicon Nanoinjection Lance Array |url=https://asmedigitalcollection.asme.org/nanoengineeringmedical/article/doi/10.1115/1.4028603/374732/Injection-of-Propidium-Iodide-into-HeLa-Cells |journal=Journal of Nanotechnology in Engineering and Medicine |language=en |volume=5 |issue=2 |article-number=021008 |doi=10.1115/1.4028603 |issn=1949-2944|url-access=subscription }}]
* '''2023''' - Researchers used nanoinjections and low voltages to add [siRNA](/source/siRNA) to cells and knockdown [gene expression](/source/gene_expression). They found that they could knock down the gene expression of [TRIOBP](/source/TRIOBP) in indiviadual cells by about 40%.[{{Cite journal |last1=Shokouhi |first1=Ali-Reza |last2=Chen |first2=Yaping |last3=Yoh |first3=Hao Zhe |last4=Murayama |first4=Takahide |last5=Suu |first5=Koukou |last6=Morikawa |first6=Yasuhiro |last7=Brenker |first7=Jason |last8=Alan |first8=Tuncay |last9=Voelcker |first9=Nicolas H. |last10=Elnathan |first10=Roey |date=2023-08-17 |title=Electroactive nanoinjection platform for intracellular delivery and gene silencing |journal=Journal of Nanobiotechnology |language=en |volume=21 |issue=1 |pages=273 |doi=10.1186/s12951-023-02056-1 |doi-access=free |issn=1477-3155 |pmc=10433684 |pmid=37592297}}]
* '''2024 -''' Researchers explored the effects of different shaped lance tips and protocols to manufacture [CAR-T cells](/source/CAR_T_cell). They found significant delivery differences between nanolance shapes and voltage combinations depending on payload type, emphasizing a need for diliberate choice in shape selection when nanoinjections are performed.[{{Cite journal |last1=Chen |first1=Yaping |last2=Shokouhi |first2=Ali-Reza |last3=Voelcker |first3=Nicolas H. |last4=Elnathan |first4=Roey |date=2024-06-18 |title=Nanoinjection: A Platform for Innovation in Ex Vivo Cell Engineering |url=https://doi.org/10.1021/acs.accounts.4c00190 |journal=Accounts of Chemical Research |volume=57 |issue=12 |pages=1722–1735 |doi=10.1021/acs.accounts.4c00190 |issn=0001-4842 |pmc=11191407 |pmid=38819691}}]
== Fabrication ==
The lance is made using the [polyMUMPs](/source/polyMUMPs) fabrication technology. It creates a gold layer, and two structural layers that are 2.0 and 1.5 μm thick respectively. It is a simple process, which makes it good as a platform to prototype [polysilicon](/source/Polycrystalline_silicon) [MEMS](/source/Microelectromechanical_systems) devices at a low commercial cost of fabrication. The lance has a solid, tapered body, that is 2 μm thick, with a tip width of 150 nm. The taper is set at 7.9°, coming to a maximum width of 11 μm. Two highly folded electrical connections provide an electrical path between the lance and two equivalent bond pads, with a gold wire connecting one of the bond pads to an [integrated circuit](/source/integrated_circuit) chip carrier's pin. The carrier is then placed into a custom built electrical socket.[{{Cite journal|last1=Aten|first1=Q. T.|last2=Jensen|first2=B. D.|last3=Burnett|first3=S. H.|last4=Howell|first4=L. L.|date=December 2011|title=Electrostatic Accumulation and Release of DNA Using a Micromachined Lance|journal=Journal of Microelectromechanical Systems|volume=20|issue=6|pages=1449–1461|doi=10.1109/JMEMS.2011.2167658|bibcode=2011JMemS..20.1449A |s2cid=59961 |issn=1057-7157}}]
In the situation of fertilizing eggs, the lance is incorporated into a kinematic mechanism consisting of a change-point parallel-guiding six-bar mechanism and a compliant parallel-guiding folded-beam suspension.{{Citation needed|date=October 2025}}
== Techniques ==
=== Electrophoretic Injection ===
[Electrophoretic](/source/Electrophoresis) injection remains the most common form of nanoinjection. Just as with the other methods, a lance ten times smaller than that of microinjection is used. Preparing the lance for injection, a positive charge is applied, attracting the negatively-charged DNA to its tip. After the lance has reached a desired depth within the cell, the charge is reversed, repelling the DNA into the cell.[{{Cite journal|last1=Lindstrom|first1=Zachary K.|last2=Brewer|first2=Steven J.|last3=Ferguson|first3=Melanie A.|last4=Burnett|first4=Sandra H.|last5=Jensen|first5=Brian D.|date=2014-10-03|title=Injection of Propidium Iodide into HeLa Cells Using a Silicon Nanoinjection Lance Array|journal=Journal of Nanotechnology in Engineering and Medicine|volume=5|issue=2|pages=021008–021008–7|doi=10.1115/1.4028603|s2cid=135872805 |issn=1949-2944}}]
== Applications ==
By delivering certain particles into cells, [disease](/source/disease)s can be treated or even cured. [Gene therapy](/source/Gene_therapy) is possibly the most common field of foreign material delivery into cells and has great implications for curing human genetic diseases.
For example, in a recent experiment two monkeys [colorblind](/source/colorblind) from birth were given gene therapy treatment via microinjection. As a result of gene therapy, both animals had their color vision restored with no apparent side effects.[{{Cite journal |last1=Mancuso |first1=Katherine |last2=Hauswirth |first2=William W. |last3=Li |first3=Qiuhong |last4=Connor |first4=Thomas B. |last5=Kuchenbecker |first5=James A. |last6=Mauck |first6=Matthew C. |last7=Neitz |first7=Jay |last8=Neitz |first8=Maureen |date=2009-10-08 |title=Gene therapy for red-green colour blindness in adult primates |journal=Nature |volume=461 |issue=7265 |pages=784–787 |doi=10.1038/nature08401 |issn=1476-4687 |pmc=2782927 |pmid=19759534 |bibcode=2009Natur.461..784M }}] Traditionally, gene therapy has been divided into two categories: biological ([viral](/source/virus)) vectors and chemical or physical (nonviral) approaches. Although viral vectors are currently the most effective approach to delivering DNA into cells, they have certain limitations, including [immunogenicity](/source/immunogenicity), [toxicity](/source/toxicity), and limited capacity to carry DNA.[{{Cite journal|date=2005-04-05|title=Physical methods for gene transfer: Improving the kinetics of gene delivery into cells|journal=Advanced Drug Delivery Reviews|language=en|volume=57|issue=5|pages=733–753|doi=10.1016/j.addr.2004.12.007|issn=0169-409X|last1=Mehierhumbert|first1=S.|last2=Guy|first2=R.|pmid=15757758 |bibcode=2005ADDR...57..733M }}]
== Alternatives ==
Microinjection is the predecessor to nanoinjection. Still used in biological research, microinjection is useful in the examination of non-living cells or in cases where cell viability does not matter. Using a glass pipette 0.5-1.0 micrometers in diameter, the cell has its membrane damaged upon puncture. As opposed to nanoinjection, microinjection uses DNA-filled liquid driven into the cell under pressure. Depending on factors such as the skill of the operator, survival rates of cells undergoing this procedure can be as high as 56% or as low as 9%.