surface ligands determine , to a great extent , the biologically relevant functions of various nanosized particles .
this is particularly important for drug delivery , where active ligands provide specific interactions to guide particles to the desired place in the body , and for biosensing , where moieties that recognize the analyte of choice provide the necessary selectivity in binding the analyte to the sensor surface .
metal  organic frameworks ( mofs ) , also known as coordination polymers , are a relatively new class of crystalline porous materials that are formed by the assembly of metal ions with rigid rodlike organic ligands in suitable solvents.1 mofs offer ultrahigh porosity , large internal surfacetovolume ratios , tunable structures , and tailorable chemistry.2 mofs have attracted considerable interest in many potential applications.3 in particular , the use of mofs in biomedicine is growing rapidly because of their advantages in drug delivery and imaging applications over traditional carriers such as organic polymers4 and inorganic materials.5 therefore , surface modification strategies are important for tuning the properties of mofs toward specific applications , such as drug delivery and biosensing .
mofs can be used in the development of multifunctional nanocarriers for biomedicine , because they can be tailored in order to have : ( i )  a particle size appropriate for intravenous applications,6 ( ii )  a specific trigger for assembly and disassembly that allows controlled drug encapsulation and release,3f , 7 ( iii )  the ability to serve as a contrast agent8 ( e.g. , to differentiate between normal and cancerous cells ) , ( iv )  a surface functionalized with stabilizers6 , 9 ( such as peg ) to permit circulation in the bloodstream for a prolonged period of time , and ( v )  celltargeting surface moieties to allow targeted drug delivery.8b , 10 
  in particular , mil88a has been used for drug encapsulation and as a contrast agent by horcajada , gref , and coworkers.6 mil88a has many advantages compared to other mofs because of its flexible framework , low toxicity , endogenous degradation products ( iron and fumaric acid ) , and easy synthesis in an aqueous medium , resulting in beneficial properties such as a good biocompatibility , degradability , imaging properties , and high loading capacities .
recently , dna moieties have been incorporated on mof surfaces to allow particle stabilization , dna detection , and cellular entry.11 mirkin et  al.11a created the first nucleic acid  mof nanoparticle hybrids , in which the oligonucleotides were appended with a dibenzylcyclooctyne containing linker to an azidefunctionalized uio66 mof ; these were used to create a steric and electrostatic barrier that stabilize the particles in highly dielectric media and at the same time allows them to enter cells .
moreover , nanosized mofs ( nanomofs ) were noncovalently linked to fluorescently labeled pna for mirna detection.12 
  many researchers used mofs as sensing platforms for dna detection using electrostatic interactions and  stacking .
chen et  al.11b developed a selective fluorescence sensor for sequencespecific recognition of hiv dsdna in vitro , using a mof functionalized with triplexforming oligonucleotide ( tfo ) probes as the sensing platform . using a similar approach , li
et  al.11c introduced mil101 as a fluorescence anisotropy ( fa ) amplifier for dna detection .
this mof provided a strong affinity for negatively charged ssdna through  stacking and electrostatic interactions .
jiang et  al.11d used a similar approach but using uio66nh2 instead of mil101 . here
, dna was detected by electrostatic interactions and  stacking between ssdna and the benzylamino group in uio66nh2 .
the authors developed a platform that is capable of distinguishing complementary and mismatched target sequences with high sensitivity and selectivity .
kang et  al.12 used this concept for sensing multiplexed mirnas in living cancer cells by a peptide nucleic acid labeled with a fluorophore adsorbed on a nanomof .
previously our group developed a strategy to simultaneously control both the size and the surface functionalization of mof particles.13 mil88a was synthesized and its size was tuned by varying the stoichiometric ratio between multivalent and monovalent ligands . at the same time , surface functionalization was successfully accomplished and the surface coverage was quantified using a fluorinecontaining capping ligand .
moreover , surface functionalization with peg and biotin groups was shown , and the latter was bound to fluorescently labeled streptavidin . here
, we describe the development of general surface functionalization strategies of mofs , using both covalent and noncovalent approaches , based on the concept of simultaneous particle formation and surface functionalization .
the noncovalent strategy is applied to the formation of peptide nucleic acid ( pna)functionalized mil88a , which is used subsequently for the selective binding of dna at the mof surface .
flow cytometry is used to analyze sufficiently large numbers of particles so that differences in hybridization efficiency can be detected with statistical significance .
native mil88a was synthesized using ferric chloride ( as the metal ion providing the secondary building unit ) and fumaric acid ( as the organic linker ) providing a final structure of fe3o(oocc2h2coo)3clz3  h2o . using a previously described methodology,13 mil88a
mil88a ) by replacing a small fraction of the organic linker fumaric acid with 10undecynoic acid ( 5  % ) or biotin  cooh ( 1  % ) , respectively , as shown in scheme  1  a. powder xray diffraction ( xrd ) ( see figure  s1 in the supporting information ) showed that all mofs were semicrystalline .
the changes observed in the spectra of the functionalized mofs compared to the native mil88a are attributed to the flexibility of the framework and the effect of water absorption into the pores.13 , 14 , 15 the presence of the ligands may further affect the degree of hydration .
figure  1 shows scanning electron microscopy ( sem ) images and the average length and width of mil88a , alkynemil88a , and biotinmil88a particles , which confirm the successful synthesis of the mofs . as observed before,13 , 15 adding smallmw ligands to the mof surface has only limited effect on the mof particle size . here
, we only observe a significant decrease in the width but not the length of the particles . because functionalization was our primary target here
a )  synthesis of mil88a functionalized with a capping ligand ; b )  covalent surface functionalization by the click reaction between alkyne  mil88a and azide  coumarin ; c )  noncovalent surface interactions between biotin 
mil88a , af488sav , and biotin  pna ; d )  chemical structures of the compounds used here .
sem images of a )  mil88a and mil88a functionalized with b )  5  % 10undecynoic acid and c )  1  % biotin  cooh capping ligand ; d )  average particle dimensions from sem images  ( a  c ) ; length ( dark gray ) and width ( light gray ) .
teller ( bet ) surface area , from 347  m  g for unfunctionalized mil88a to 13.4  m  g for biotinmil88a .
this strong decrease is consistent with earlier observations.2b , 13 , 15 given the similar molecular weight of undecynoic acid compared to the fluorinecontaining capping ligand described before,13 we presume the bet areas to be similar .
the zeta potential values of uncoated mil88a ( 19.43.4  mv ) were shifted to more neutral values of 12.40.3 and 12.40.4  mv in the case of alkynemil88a and biotinmil88a , respectively , in line with a partial capping of free fe coordination sites at the surface , as discussed before.15 in summary , we successfully functionalized the surface of mil88a with alkyne and biotin groups , which can be used for further covalent and noncovalent functionalization , respectively .
covalent surface functionalization was achieved by employing the cucatalyzed click reaction at the surface of alkynemil88a ( see scheme  1  b ) using 3azido7hydroxycoumarin as reagent , resulting in coumarin 
the reaction was carried out by adding an excess of azide , using tetrakis(acetonitrile)copper(i ) hexafluorophosphate as catalyst , in the presence of tris(benzyltriazolylmethyl)amine ( tbta ) , followed by continuous stirring overnight at room temperature .
after the click reaction , bright field and fluorescence microscopy images ( figure  2 ) were taken .
the images show mof particles with a high fluorescence intensity , demonstrating the successful functionalization of the surface of mil88a . performing the click reaction at approximately twofold higher concentrations of both alkyne and azide ( data not shown ) led to a small increase of fluorescence intensity .
control experiments were performed with a mof lacking the alkyne function ( control  1 ) , or with alkyne mof but in the absence of the copper catalyst ( control  2 ) .
the bright field and fluorescence images of these controls ( figure  s2 in the supporting information ) indicate the absence of fluorescence in both cases , which confirms the need of alkyne groups on the mof surface and the presence of the catalyst to achieve the successful click synthesis . in summary , these results demonstrate the specific covalent surface functionalization of mil88a using the cucatalyzed click reaction . 
  bright field  ( a ) and fluorescence  ( b ) microscopy images of mil88a after reaction of alkyne 
mil88a ( 0.49  mm of alkyne ) with coumarin azide ( 3.3  mm ) . to demonstrate the feasibility of employing mil88a as a platform for pna / dna functionalization for binding target dna , we chose to functionalize mil88a with pna with a specific sequence that has already been tested in sensing applications.16 three different dna targets were chosen to evaluate the binding selectivity of the mof  pna system : the full match ( dnafm ) strand , a singlebase mismatched ( dna1 mm ) strand , and a randomized sequence ( dnarand ) ( scheme  2 ) .
all dna targets were labelled with cy5 for visualization . to bind biotinylated pna to the mof platform for the selective binding of dna ( scheme  2  a ) , we employed the specific noncovalent interaction between biotinmil88a and alexafluor488labeled streptavidin ( af488sav ) ( scheme  1  c).13 
  a )  noncovalent surface interactions between biotin 
mil88a , af488sav , and biotin  pna and further hybridization with dna and b )  structures of the oligonucleotides used here .
as shown in scheme  2  a , af488sav was first attached to biotin 
mil88a using a high sav / biotin ratio of 15 to suppress interparticle crosslinking that was observed before at lower ratios.13 the fluorescence intensities of af488sav and cy5 ( dna targets ) were measured using flow cytometry , as shown in figure  3 and figure  4 .
mil88a particles in the absence of any dyes were below 10 for emission of both dyes ( figure  3  a ) . for biotin 
mil88a conjugated with af488sav , significantly higher intensities were observed for the emission of af488 ( figure  3  b ) confirming the conjugation between biotin  mil88a and af488sav . to focus on the dnarecognition properties of the particles conjugated with af488sav
, we selected in all cases those particles that passed the minimum relative fluorescence intensity of 10 for the af488 emission ( figure  3  c ) .
this enables easier visualization of differences in selectivity for the different cy5labeled dna target probes used here .
the few events above the relative fluorescence cy5 intensity of 10 observed in figure  3  c ( purple markers ) are attributed to background noise because cy5 is not present in this sample . for the pna 
dna hybridization , we used a relative fluorescence intensity staining of 10 for cy5 and used the same gate settings for both dyes at all experiments described below . 
 
flow cytometry results for : a )  biotin  mil88a , and b )  the conjugate of biotin 
mil88a and af488sav , and c )  the fraction of particles of b for which the af488 intensity is above 100 .
the blue regions indicate the fractions of particles for which the af488 intensity is below 100 , green indicates the fractions of particles for which the af488 intensity is above 100 ( indicative of conjugation between biotin  mil88a and af488sav ) , and pink represents the fractions of particles for which both the af488 and cy5 intensities are above 100 .
flow cytometry  ( a ,  c ,  e ,  g ) and confocal microscopy  ( b ,  d ,  f ,  h ) results for biotin  mil88a particles after incubation with af488sav followed by incubation with either dnafm  ( a ,  b ) or biotin  pna followed by hybridization with dnafm  ( c ,  d ) , dna1 mm  ( e ,  f ) , and dnarand  ( g ,  h ) after washing at 40  c . in the flow cytometry graphs , only the fractions of particles are shown for which the af488 intensity passed the relative fluorescence intensity of 100 .
the green regions indicate particles with a high intensity of af488 and pink represents a high intensity for both af488sav and cy5 . in the confocal images  ( b ,  d ,  f ,  h )
, the top left panel corresponds to af488sav , and top right corresponds to cy5 ( marker bound to dna ) fluorescence channels , bottom left is the microscope bright field image , and bottom right is the overlay .
mil88a , preconjugated with af488sav , were incubated with biotin  pna at room temperature using a pna / sav ratio of 3 .
thereafter , hybridization between pna and the complementary dnafm target , labeled with cy5 , was performed at 30  c for 1  h using a pna / dna ratio of 1 , followed by washing at 40  c .
washing at room temperature instead gave nonspecific electrostatic interactions between the mof particles and the dna targets ( figure  s3 in the supporting information ) .
the same hybridization and washing procedure was followed for the dna1 mm and dnarand targets .
the samples were characterized using flow cytometry and confocal microscopy . in the absence of biotin  pna , incubation with dnafm did not yield cy5 fluorescence for the particles ( figure  4  a ,  b ) .
in contrast , in the presence of pna , incubation with dnafm gave colocalization of high fluorescence intensities of af488 as well as cy5 ( figure  4  c ,  d ) .
these results indicate that pna and dnafm have been successfully and specifically hybridized at the mof surface .
in addition to dnafm , the singlemismatch target dna1 mm and the random dnarand target were used to investigate the selectivity of the sensing platform , likewise characterized using flow cytometry and confocal microscopy ( figure  4  d  h ) .
flow cytometry showed high cy5 intensities for the adduct of mof+pna and dna1 mm ( figure  4  e ) but low intensities for the adduct of mof+pna and dnarand ( figure  4  g ) .
the high intensity of mof+pna with dna1 mm is attributed to the high sequence similarity ( 93  % ) of dna1 mm compared to dnafm . in the case of the interaction of mof+pna with dnarand ,
the fluorescence intensity distribution ( figure  4  g ) was similar to the control without biotin  pna ( figure  4  a ) .
fluorescence imaging ( figure  4  f ,  h ) confirmed these findings by the colocalization of fluorescence of af488sav and cy5 ( figure  4  f ) for the interaction between mof+pna and dna1 mm , and a strong intensity of only af488sav ( figure  4  h ) for mof+pna with dnarand .
fluorescence intensity profiles were taken from several confocal images , as shown in figure  s4a  d ( see the supporting information ) . the ratio , r , of the intensity differences of i 
 cy5 and i 
 af488 between the peak and background intensities of cy5 and af488sav , respectively , was measured and plotted in figure  s4e for all cases .
figure  s4e shows that the hybridization of mof+pna with complementary dnafm exhibited the largest r value , followed by mof+pna with singlebase mismatch dna1 mm .
the adducts of mof ( without pna ) with dnafm and of mof+pna with dnarand show very small r values , due to very low cy5 intensities in these cases .
statistical analyses ( ttests ) , shown in table  1 , gave p values below 0.05 only for the comparisons of mof+pna with dnafm with mof ( no pna ) with dnafm and mof  pna with dnarand . in the case of mof  pna with dna1 mm , the confocal intensity ratios were not significantly different .
the main reason for this is most likely the lack of a sufficiently large number of analyzed particles . 
 
pna+dnafm vs. different cases using the average r values from figure  s4e ( confocal imaging ) and the mean intensity values from figure  5 ( facs ) .
in contrast , flow cytometry provides convenient access to the analysis of much larger numbers of particles . to analyze these results
more deeply , we compared the mean cy5 intensities ( m 
 cy5 ) of all events for each sample as well as the fractions of particles with a cy5 intensity over 10 ( table  2 , also including the total numbers of particles analyzed ) .
pna+dnafm gave the highest m 
 cy5 value ( 103 ) , followed by mof 
as expected , the values for mof , mof ( no pna)+dnafm , and mof  pna+dnarand had similar and low m 
 cy5 values .
pna+dnafm showed the largest fraction ( 46.5  % ) of particles with a cy5 intensity above 10 in comparison to all other cases , indicating higher degrees of hybridization . in the case of mof 
pna+dna1 mm ( 18.3  % ) , the highintensity fraction was still higher than for mof , mof ( no pna)+dnafm , and mof 
average intensity , m 
 cy5 , for all the events , fractions ( % ) with a cy5 intensity below or above 100 , and total numbers of particles , as measured by flow cytometry for mof ( = adduct of af488sav and biotin 
mil88a ) , mof  pna+dnafm , mof ( no pna)+dnafm , mof  pna+dna1 mm , mof  pna+dnarand after washing at 40  c .
next , we determined the average cy5 intensities of only those particles with a cy5 intensity above 10 , as shown in figure  5 .
the values for mof incubated with dnafm ( in the absence of pna ; 163 ) , mof+pna with dnarand ( 148 ) are comparable to the value of mof ( 153 ) , and are based on comparably low numbers of particles because the highintensity fraction of particles is small for these cases .
pna incubated with dna1 mm , the average intensity was only slightly less , with a mean intensity value of 180 .
yet , a statistical analysis ( ttest ) of the data shown in figure  5 ( table  1 ) showed that all p values for comparisons of the adduct of mof+pna with dnafm with those of mof ( no pna ) with dnafm , mof  pna with dna1 mm , and mof  pna with dnarand were below 0.05 .
thus , we conclude that pna immobilized on mil88a is able to distinguish between complementary dnafm and singlebase mismatch dna1 mm when analyzing the data using flow cytometry , because sufficiently large numbers of particles can be analyzed simultaneously . in summary
, these results indicate that ( i )  pna is mandatory for the specific binding of dna , ( ii )  dnafm and dna1 mm are hybridized with this pna but to measurably different extents , and ( iii )  this sensing platform is able to differentiate between the fully complementary dnafm , mismatched dna1 mm , and random dnarand targets . 
 
mean fluorescence intensities as measured by flow cytometry for mof and mof  pna , upon incubation with dnafm , dna1 mm , or dnarand , after washing at 40  c .
in conclusion , we have developed versatile methods for the surface functionalization of mil88a using either covalent or noncovalent interactions . using the noncovalent approach with biotin 
we tested this wellknown ligand attachment strategy because of its easy procedure and high rate of attachment .
future work will have to show whether the covalent click chemistry can result in a similarly robust pna attachment strategy .
the pnafunctionalized mof particles were used to achieve dna binding and to assess the dna binding selectivity . by washing mofs at moderately high temperature , nonspecific adsorption between dna and the particles can be avoided .
overall , the sensing platform was able to distinguish between complementary and singlebase mismatched or random sequences .
the difference between complementary and singlebase mismatched dna was small , but statistically significant when analyzing large numbers of particles using flow cytometry .
the difference between these targets can possibly be improved further by washing at a temperature closer to the melting temperature of the dna  pna duplex . for future work
, we envisage the creation of different dna binding strategies for recognizing proteins , transcription factors , etc .
this method can in principle be used to assay different analytes by introducing probes that selectively bind to the analytes .
these features contribute to a simpler , efficient , and more general platform that could be expanded to other mofs and applications .
as a service to our authors and readers , this journal provides supporting information supplied by the authors .
such materials are peer reviewed and may be reorganized for online delivery , but are not copyedited or typeset .
technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors .
 abstracta dnasensing platform is developed by exploiting the easy surface functionalization of metal  organic framework ( mof ) particles and their highly parallelized fluorescence detection by flow cytometry . 
 two strategies were employed to functionalize the surface of mil88a , using either covalent or noncovalent interactions , resulting in alkynemodified and biotinmodified mil88a , respectively . 
 covalent surface coupling of an azidedye and the alkyne  
 mil88a was achieved by means of a click reaction . 
 noncovalent streptavidin  biotin interactions were employed to link biotin  
 pna to biotin  
 mil88a particles mediated by streptavidin . 
 characterization by confocal imaging and flow cytometry demonstrated that dna can be bound selectively to the mof surface . 
 flow cytometry provided quantitative data of the interaction with dna . making use of the large numbers of particles that can be simultaneously processed by flow cytometry , 
 this mof platform was able to discriminate between fully complementary , singlebase mismatched , and randomized dna targets . 
Introduction
Results and Discussion
Conclusion
Conflict of interest
Supporting information