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MATERIALS & TECHNOLOGY_Nanosensors

Biomolecules in Action

Techniques that provide insights into the nanoworld continue to garner Nobel Prizes. However, none of those methods has made it possible to observe exactly how and other . Frank Vollmer, Leader of a Research Group at the Max Planck Institute for the Science of Light in Erlangen, has now changed all that – with a plasmonic nanosensor.

TEXT CHRISTIAN MEIER

rank Vollmer spared no effort. an inventor. He needed a kind of cam- cules, a third reads genetic information Sure, scientists often put a lot era that can record individual enzymes − and often have moving parts. of time into developing new and the movements of their parts, but An that carries out a funda- methods, especially when the laws of physics appeared to prohib- mental process reveals just how they’re looking for something it such a device. Nevertheless, the sci- similarly biological and technological fundamentallyF new. But Vollmer, a bio- entist recently caught a glimpse of one tools function: DNA dupli- chemist by training, spent around 20 of the most delicate mechanisms of life. cates the DNA , thus enabling years working as a physicist to devise cells to divide. This process underlies the tool he needed to realize his proj- BIOLOGICAL TOOLS WORK LIKE every form of . The en- ect. Ever since his days as a biochemis- TECHNOLOGICAL ONES zyme is reminiscent of a hand with a try student in Hanover in the 1990s, thumb and fingers that literally grasps he’s always wanted to watch the ma- “We all consist of ,” says the DNA strand being copied. chinery of life at work. Vollmer. That’s true. For one thing, pro- Observing enzymes such as DNA That desire still drives him today in teins, enzymes and other biomolecules polymerase in action is especially im- his role as a Research Group Leader at are just a few nanometers in size. One portant when the biological machines the Max Planck Institute for the Science nanometer, a millionth of a millimeter, develop a hitch. Catching a glimpse of of Light in Erlangen and a professor at is as tiny as a soccer ball is in relation to these nanomachines at work could the University of Exeter. He wants to planet Earth. For another thing, like then reveal where the problem lies. understand why the tiny machines that man-made machines, these tiny work- When DNA is copied during divi- keep life processes running sometimes horses in the body each serve a specific sion, errors can occur, much like typo- go awry, causing us to become ill. To re- purpose − one transports oxy- graphical errors in a book. In some cir-

alize his plan, Vollmer had to become gen, another breaks down sugar mole- cumstances, such mutations can lead to Graphic: Han and Holm, based on original material from the MPI for Science of Light

64 MaxPlanckResearch 1 | 18 Graphic: Han and Holm, based on original material from the MPI for the Science of Light fluorescent , or markers. The cell componentsthatarelabeled with resolution limit, theycanonly observe py, use physical tricks to get around this some methods,suchasSTED microsco around 200nanometers.Although or light’s wavelength is about the limit, light. Afocalpointmeasuringhalfthe focused toasmallenoughpointof because visible light can’t be structures croscope isunabletoresolvenano light isapracticallyneutralobserver. heating ofasample.Inshort,visible little cules. In addition, they cause very energy can virtually pulverize mole gentler than,say, X-rays,whosehigh magnetic spectrum. They are much waves areaharmlesspartoftheelectro cameras andvideodo.Light uses visiblelight–justlikeoureyes, nanotechnology atwork.To dothis,he lmer foundawaytowatchnature’s rapid nature of life processes, Frank Vol- matters either.” rapidlydoesn’tand closesvery help laments. “Thefactthatthehandopens our understanding of disease,” he thingthat’ssee thevery socrucialfor movements ofbiomolecules.“We can’t has sofarbeenlargelyblindtothe lose theirabilitytofunction. change shape,causingbiomoleculesto nesses, suchasAlzheimer’s, diseases, suchascancer. Inotherill- the enzyme can be observed. be can enzyme the of movement the way, this In changes. spot light the with overlap the tightly, light focuses that nanowire gold a to bound is the When genome. grasps the building blocks of our openingand closing, DNA polymerase hand a Like grip: a gets enzyme An Unfortunately, anormallightmi Despite thetinysizeandextremely Frank Vollmer regretsthatscience 1 | MaxPlanckResearch 18MaxPlanckResearch 65 - - - - - MATERIALS & TECHNOLOGY_Nanosensors

Frank Vollmer wanted to understand how RNA is read. » To answer this question, he first had to develop a suitable method.

points of fluorescent light then show the curvature instead of spreading out New York. He shone laser light into a how the biomolecules move about, in all directions. Similarly, a light wave glass bead and used it to detect pro- much as car headlights at night reveal runs along the inside of a glass bead teins. At the time, though, he was able where a car is heading. But a great deal measuring just a few micrometers to detect only multiple proteins bound remains in the dark, such as whether (thousandths of a millimeter), whizzing to the bead, as only then did the wave- the points of light belong to a car or a around inside it tens of thousands of length of the circulating light change truck, or whether a door is opened on times. As it circulates, the wave keeps appreciably. However, to understand a stationary car. In the same way, the encountering itself. At a well-defined how the nanomachines of life work, it fluorescent molecules don’t show in de- wavelength that depends on the size of is necessary to observe single biomole- tail whether or how a protein changes the glass bead, the wave crests travel- cules. An ensemble of proteins produc- shape. Moreover, scientists can never ling around the glass bead overlap pre- es only an average value from which it be sure that the labels don’t interfere cisely with the crests of previous laps. is impossible to determine the behav- with the function of the biomolecule Physicists refer to this phenomenon as ior of individual molecules. they are investigating. resonance. This is analogous to the res- onant body of a musical instrument THE TRICK: A COMBINATION OF LIKE A WHISPERING GALLERY that, because of its dimensions, ampli- OPTICS AND PLASMONICS fies only certain sounds. Frank Vollmer reached the limits of op- The optical whispering-gallery ef- While head of a research group at Har- tical observation at the latest during his fect makes the glass bead a very sensi- vard University, Vollmer thus worked doctoral work at Rockefeller University tive sensor. The light wave extends on refining the method. He ultimately in New York, where he was researching somewhat beyond the surface of the succeeded in using the glass bead to de- how the second carrier of genetic infor- bead. If a virus or protein, for example, tect single viruses. It was a remarkable mation, RNA, is read. “At that point, I adheres to the surface of the ,­ triumph, but he had not yet achieved realized that a suitable method had yet the particle interacts with the light the actual goal of observing single bio- to be invented.” The decid- wave, slowing it down a little. The ef- molecules. After all, a virus is still ten ed to devise one himself and became a fect is the equivalent of increasing the times larger than a protein. physicist to do it. He had, after all, al- circumference of the bead’s wall. Con- “We soon realized that it would be ways been interested in this discipline. sequently, the virus or protein alters the far more difficult to detect individual Before long, Vollmer came across a resonant wavelength of the microbead. biomolecules,” Vollmer says. The light technique that pointed in the right di- Although the change is minimal, it can path changes in relation to the volume rection. It is the optical analogue of a be detected thanks to the sharpness of of a detected . The change whispering gallery. In a rotunda, a word the resonance. caused by a single molecule is therefore whispered toward the wall can easily be Vollmer succeeded in using the op- only about one-thousandth the magni- heard on the opposite side of the cham- tical whispering gallery as a biosensor tude of that caused by a virus, putting ber because sound waves travel along for the first time while still working in it well below the detection limit. Fellow

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physicists advised Vollmer to try other can interact with light was also crucial An optical whispering gallery as a nano - methods. “But such methods would for Vollmer’s plans. The researchers rea- sensor: When laser light of a suitable have required much more complicated soned that if they attached a nanowire color (here red light) is guided through a glass fiber into a glass microbead, it experimental setups,” Vollmer explains. about the size of a single biomolecule circulates within the bead like sound in He wanted a tool that can use to an optical whispering gallery, the a rotunda (1). Gold nanowires attached relatively easily. light waves that extend beyond the sur- to the bead concentrate the light by In 2010 he came up with an idea face would excite plasmons in the met- means of a plasmonic effect. Proteins and other molecules that bind to the that would lead to a breakthrough. The al. That, in turn, would generate light nanowires (2) change the wavelength trick was to combine optics with a very waves on the surface of the wire. of the light coupled to the bead, thus different branch of physics: plasmon- allowing the molecules and their ics. Plasmonics exploits the fact that A HOTSPOT AMPLIFIES movements to be detected. Such electrons in a metal form a sort of col- THE INTERACTION nanosensors for proteins and their movements were successfully tested lective, similar to a liquid, that moves for the first time in an experimental freely past the firmly fixed atomic cores Thus, a nanowire would effectively draw setup at the Max Planck Institute for in the crystal lattice. Because the nega- the light wave out of the glass bead and the Science of Light (3). tively charged electrons are attracted to focus it on a spot about the size of a the positively charged atomic cores as protein. This hotspot focusing, as Voll- though they were attached to them by mer calls it, would amplify the interac- coil springs, the collective oscillates tion between light and the object un- back and forth. The oscillations of the der investigation. In this way, even a electric charge are called plasmons. single biomolecule could lengthen the Plasmons produce an electrical path of the light to the extent that the wave that extends beyond the metal change would be measurable. surface, just like light waves extend be- Vollmer set about implementing yond the surface of the optical whisper- the idea at the Max Planck Institute for

Graphic: Frank Vollmer/Science Advances (left), Steven Haywood (right) ing gallery. The fact that such plasmons the Science of Light in Erlangen. DNA

1 | 18 MaxPlanckResearch 67 MATERIALS & TECHNOLOGY_Nanosensors

A nanowire draws the light wave out of the glass bead » and concentrates it on a spot the size of a protein.

polymerase served as a good test en- In another experiment, the researchers After this success, the researchers won- zyme because its opening and closing attached the polymerase to the nano­ dered about the limits of their new motions resembling those of a “hand” wire and mixed DNA into the solution. method. To their own astonishment, should result in a periodic increase and Schuldes had previously confirmed they were even able to observe single decrease of the overlap between the en- that attaching the enzyme to the gold ions – that is, electrically charged at- zyme and the light at the hotspot, doesn’t affect its activity. In both cas- oms. The zinc and mercury ions they much like the shadow of a hand chang- es, during the copying process, the used are one hundred times smaller in es as it opens and closes in front of a polymerase was inside the light spot diameter than a protein. It helped the light source. focused by the nanowire and, it was researchers that the gold nanowires ta- First, Isabel Schuldes, a master’s de- hoped, would change the measure- pered to a single gold at the tip. gree student in Frank Vollmer’s group, ment signal as the enzyme hand opened The plasmonic light spot therefore fo- attached gold nanowires with a diame- and closed. cuses on an extremely small spot at the ter of just 10 nanometers and a length very tip, causing the electrons to gain of 40 nanometers to a glass bead with THE RESEARCHERS EVEN more energy than they would normal- a diameter of 80 microns. Normally, OBSERVED SINGLE IONS ly have in gold. Because of their energy electrostatic forces would bind the gold gain, the charge carriers activate a reac- to the glass. In Erlangen, however, this And the researchers did indeed observe tion between the gold and the mechanism conflicted with the chemi- their signal waxing and waning in cy- mercury ions. The researchers in Erlan- cal conditions required by the poly- cles of about 20 to 50 thousandths of a gen were able to observe this reaction. merase. Its preferred pH tended to break second. “For the first time, we were able “It’s not about detecting single mer- the electrostatic bond. After an elabo- to observe protein dynamics without cury ions,” Vollmer stresses. The sensi- rate search, Schuldes found a linker using markers,” says Vollmer. And they tivity for single ions can be used, for ex- molecule that binds the nano­wire to were able to do so live, in such condi- ample, to investigate the function of the glass bead: “That gave me a great tions as occur in nature. Vollmer even ion channels, he says. These channels sense of achievement,” she says. thinks it is possible to record the se- are embedded in the membranes of Now the scientists were able to fo- quence of the DNA letters as the poly- neural cells, for instance, and help con- cus their nanospotlight on biological merase is reading them. Copying errors duct electrical stimuli through nerves. machines. They attached DNA strands could be detected this way, too. “It Thanks to its ability to track reac- to the fine wires and dipped their nano- would also be a very simple and inex- tions such as that between gold atoms sensor into a polymerase solution. The pensive method for studying DNA,” and mercury ions, the nanosensor is enzyme then copied the attached DNA. Vollmer says. also suitable as a chemical tool, the bio-

68 MaxPlanckResearch 1 | 18 Top Isabel Schuldes, Ying-Jen Chen and Frank Vollmer (left to right) have made the plasmon- ic nanosensor so sensitive that it can even detect substances that form during cell death. It takes a fine touch to couple the laser light to the sensor. Bottom The nanosensor is also suitable for analyzing chemical reactions. The Max Planck researchers used it, for example, to show that molecules with amino groups (pink) adhere to gold atoms protruding from the surface of the nanowire. Molecules with thiol groups (blue), in contrast, bind to gold atoms embedded in the surface.

physicist explains. It can be used to test and optimize conditions to control re- actions to a certain extent. The team in Erlangen even showed how to do this. By increasing the light intensity in the focused light spot, they caused the elec- trons in the gold tip to reach a very high energy level. This allows a partic- ularly strong type of chemical bond, known as a , to form. In this way, the researchers created an amalgam between gold and mercury as if by turning a light dimmer switch. In an experiment with two types of molecules, the researchers demonstrat- ed the precision with which the mech- anisms of chemical reactions can be ob- served using the Erlangen method. One type of molecule bonded with the gold via an group, the other via a thi- ol group. “It turned out that the two groups react with the gold surface through two different mechanisms,” Vollmer explains. Whereas the combine only with gold atoms that protrude above the surface, the thiols bind only to atoms that are fully em- bedded in the surface. The extreme sensitivity of the nano- sensor that Vollmer’s team developed

Photo: Tilman Weishart; graphic: Frank Vollmer, design by Ella Maru Studio/Advanced Materials isn’t its only distinguishing feature. As MATERIALS & TECHNOLOGY_Nanosensors

A sensor with many senses: At the an added benefit, even non-physicists protein cytochrome c. To do this, Chen Living Systems Institute of the can use it without the need for costly coated the sensor with an that University of Exeter, Hsin-Yu Wu, Serge Vincent, Jolly Xavier, Frank special equipment, Vollmer points out. binds exclusively to cytochrome c. Vollmer, Tom Constant and It also works in aqueous milieus, giving Sivaraman Subramanian (left to biologists and physicians an eye, as it CHIPS FOR RAPID CANCER right) built an experimental setup were, that they can place right at the DRUG SCREENING that combines various approaches: center of microbiological life. the effect of an optical whispering gallery in a glass bead, the plasmonic Vollmer’s doctoral student Ying-Jen Chen is also trying to shrink the mi- concentration of the light that Chen, for example, uses the sensor to crobead method down to chip size. In- circulates inside the bead, and observe the death struggle of cells. Al- dividual cells are held in tiny channels microscopic and spectroscopic though this process, called apoptosis, is in plastic tiles barely larger than a fin- methods. The researchers’ aim is to analyze the movements of biomole- an intracellular process, Chen managed gernail. A toxin flows through the cules in as much detail as possible. to observe it from outside the cell with- channels, causing the cells to initiate out influencing the process itself. Using apoptosis. The released cytochrome c is the microbead method, Chen detected then flushed out and sent to the sensor

a chemical marker of cell death: the setup, which still occupies most of the Photo: Steven Haywood

70 MaxPlanckResearch 1 | 18 Photo: Steven Haywood ting, namelyattheLivingSystemsIn sensor inabiologicalandmedicalset now wantstousetheplasmonicnano- sion intotheworldofphysics.He to returnbiologyafterhisexcur in his research career where he is able microchannels ormechanisms. connecting cellculturesviacomplex chips thatsimulateentireorgansby on-a-chip,” Vollmer adds,referringto method issuitableforuseasanorgan- tosis incancercellstokillthem.“Our says. Manyofthosedrugstriggerapop- than isnormallypossibletoday,” Chen ple, to screen cancer drugs much faster tus sothatit,too,fitsonachip. ever, istoshrinkthemeasuringappara surface ofalabbench.Thegoal,how-

l l l POINT TO THE controlled manner. a controlled in emission the of part extinguishing by limitation this circumvents microscope area an to so light, measuring the of wavelength around the half 200than nanometerstightly more no across.focused spot be light can the A limit, STED (stimulateddiffraction emission the depletion) to According laser. focused a with excited are nano-object a on markers microscopy, fluorescent fluorescence In microscopy: STED light. with excited be can they and plasmons, as known are These Plasmon: DNA polymerase: GLOSSARY Vollmer hassincereachedapoint “The chipscouldbeused,forexam-

with gold atoms in the nanowire. parts. their of movements the record to even and action in molecules biological th polymerase, DNA of movements the track to sensor the used have researchers The to attached nanowire agold with microbead aglass of consists nanosensor The na aplasmonic developed have Vollmer Frank with working researchers Planck Max moves, the wavelength of the light coupled to the glass bead changes. bead glass the to coupled light the of wavelength the moves, nanoparticle abound or wire gold the to binds molecule abiological Whenever wide. nanometers afew just apoint on light the focus to plasmons uses it time, same the At times. of thousands of tens bead the inside circulating wavelength it . It exploits the effect of an optical whispering gallery, with light of a defined adefined of light with gallery, whispering optical an of effect the exploits . It e process of cell death, and reactions of mercury ions and organic molecules molecules organic and ions mercury of reactions and death, cell of e process nosensor that makes it possible for the first time to observe enzymes and other other and enzymes observe to time first the for possible it makes that nosensor Oscillations of the electrons in a metal can produce electrical waves. waves. electrical produce can metal a in electrons the of Oscillations An enzyme that makes a copy of DNA during . cell during DNA of copy a makes that enzyme An - - - - lights …cameraaction! movies ofbiomolecularmachines– says. He may soon be recording entire the nanotechnology of life,” Vollmer “This couldfinallyrevealthesecretsof at severalmillionframespersecond. curate 3-Datomicimagefromthedata atom byandreconstructanac- should be possible to scan molecules Using multiplefocusedlightspots,it a laserscannerforsinglemolecules. further developthenanosensor – into biomolecules tovibrate. teins byusinglighttostimulatethe sible tovisualizesuchdefectivepro- logically deformedproteins.Itispos- There, heplanstoinvestigatepatho- “This istheidealplaceforit,”hesays. stitute attheUniversityofExeter.

But FrankVollmer alsowantsto

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